03_MIB_TMT

C Ryan Miller Lab @ UAB

This R Markdown (RMD) file performs exploratory data analysis (EDA) and differential expression (DE) analysis for the 2025 Lin manuscript.

Script Timing Start

ptm <- proc.time()

Variables

# adjusted pvalue
qval <- 0.05
pval <- 0.05
lfc <- .5

# Use max of 4 cores if available.
ncore <- ifelse((detectCores()-2)>=4,4,detectCores()-2)

Set.Seed

# This will set the random number generator
set.seed(888)

MIB-MS dynamic data

Directories

# output directory
MIB_output <- paste0(Data, "Output/MIB-DYN/")
dir.create(paste0(MIB_output))

# Graphs
MIB_graphs <- paste0(Data, "Graphs/MIB-DYN/")
dir.create(paste0(MIB_graphs))

TMT protein groups Import

Import protein groups data

# TMT1 txt file was regenerated from the excel. Saved as a tab delimited text file

path <- "/Data/Input/MIB-MS/Dynamic/TMT_proteinGroups"

filelist <- list.files(path=path, full.names = TRUE)

# Import protein groups. Renames by base filename using string extraction
proteinGroups_ls <- list()
for (i in seq_along(filelist)){
  proteinGroups_ls[[sapply(strsplit(paste0(basename(filelist[i])), "_"), '[',1)]] <-
    read.table(filelist[i], sep="\t", header = TRUE )
}

# Sort alpha numeric
mixedsort(names(proteinGroups_ls), decreasing = F)

##  [1] "TMT01" "TMT02" "TMT03" "TMT04" "TMT05" "TMT06" "TMT07" "TMT08" "TMT09"
## [10] "TMT10" "TMT11" "TMT12" "TMT13" "TMT14" "TMT15" "TMT16" "TMT17" "TMT18"
## [19] "TMT19" "TMT20" "TMT21" "TMT22" "TMT23" "TMT24" "TMT25" "TMT26" "TMT27"
## [28] "TMT28" "TMT29" "TMT30" "TMT31" "TMT32" "TMT33" "TMT34" "TMT35" "TMT36"
## [37] "TMT37" "TMT38" "TMT39" "TMT40" "TMT41" "TMT42"

# Order the list of dataframe
proteinGroups_ls_ordered <- list()
for (i in seq_along(filelist)){
  proteinGroups_ls_ordered[mixedsort(names(proteinGroups_ls), decreasing = F)[i]] <- 
    proteinGroups_ls[names(proteinGroups_ls)==mixedsort(names(proteinGroups_ls), decreasing = F)[i]]
}


# Subset to kinases only
proteinGroups_kinases_ls <- list()
for (i in seq_along(proteinGroups_ls_ordered)){
 proteinGroups_kinases_ls[[names(proteinGroups_ls_ordered)[i]]] <-
   subset(proteinGroups_ls_ordered[[i]], proteinGroups_ls_ordered[[i]]$Gene.names %in% c(kinases.anno$Mouse_symbol,"EGFR"))
}

# Change EGFR to hEGFR
for (i in seq_along(proteinGroups_kinases_ls)){
  proteinGroups_kinases_ls[[i]]$Gene.names <- 
    replace(proteinGroups_kinases_ls[[i]]$Gene.name,proteinGroups_kinases_ls[[i]]$Gene.name %in% c("EGFR"),"hEGFR")
}

# Assign genes as rownames
for ( i in seq_along(proteinGroups_kinases_ls)){
  rownames(proteinGroups_kinases_ls[[i]])<- proteinGroups_kinases_ls[[i]]$Gene.names
}

# Extract out Reporter intensity
kinase_raw_counts <- list()
for ( i in seq_along(proteinGroups_kinases_ls)){
  kinase_raw_counts[[names(proteinGroups_kinases_ls)[i]]] <-
    proteinGroups_kinases_ls[[i]][,colnames(proteinGroups_kinases_ls[[i]]) %in% c("Reporter.intensity.1","Reporter.intensity.2","Reporter.intensity.3", "Reporter.intensity.4","Reporter.intensity.5","Reporter.intensity.6")]
}

# Normalization
kinase_norm <- list()
for ( i in seq_along(kinase_raw_counts)){
  kinase_norm[[names(kinase_raw_counts)[i]]]<-
    data.frame(colSums(kinase_raw_counts[[i]]))
  colnames(kinase_norm[[names(kinase_raw_counts)[i]]])<- "sums"
}

# Extract reporter 6 %. Reporter 6 is the pooled control. 
kinase_norm_per <- list()
for (i in seq_along(kinase_raw_counts)){
  kinase_norm_per[[names(kinase_raw_counts)[i]]] <-
  as.data.frame(colSums(kinase_raw_counts[[i]])[6]/sum(colSums(kinase_raw_counts[[i]]))*100)
  colnames(kinase_norm_per[[names(kinase_raw_counts)[i]]]) <- names(kinase_raw_counts)[[i]]
}

# Convert to matrix
kinase_norm_per_matrix <- kinase_norm_per %>% map( ~ .x %>%
             rownames_to_column('Reporter.intensity.6'))  %>% 
   reduce(full_join, by = 'Reporter.intensity.6') 

# Remove column label
kinase_norm_per_matrix <- kinase_norm_per_matrix[,-which(names(kinase_norm_per_matrix) %in% c("Reporter.intensity.6"))]

# Total sum of Reporter.intensity.6 %
sum(kinase_norm_per_matrix[1,])/(length(kinase_norm_per_matrix[1,]))

## [1] 5.473136

# Get a normalization factor for each TMT experiment
kinase_norm_per_matrix["norm_factor",] <- (sum(kinase_norm_per_matrix[1,])/(length(kinase_norm_per_matrix[1,])))/kinase_norm_per_matrix[1,]



colSums(kinase_raw_counts[[1]])

## Reporter.intensity.1 Reporter.intensity.2 Reporter.intensity.3 
##             35293260             37823850             31281229 
## Reporter.intensity.4 Reporter.intensity.5 Reporter.intensity.6 
##             33213317             38442283             11164385

sum(colSums(kinase_raw_counts[[1]]))

## [1] 187218323

colSums(kinase_raw_counts[[1]])[6]/sum(colSums(kinase_raw_counts[[1]]))

## Reporter.intensity.6 
##           0.05963297

colSums(kinase_raw_counts[[1]])[1]/sum(colSums(kinase_raw_counts[[1]]))

## Reporter.intensity.1 
##            0.1885139

colSums(kinase_raw_counts[[1]])[2]/sum(colSums(kinase_raw_counts[[1]]))

## Reporter.intensity.2 
##            0.2020307

# Rename the columns
kinase_raw_counts_rn <- list()
for ( i in seq_along(kinase_raw_counts)){
  kinase_raw_counts_rn[[names(kinase_raw_counts)[i]]] <- kinase_raw_counts[[i]]
    colnames(kinase_raw_counts_rn[[names(kinase_raw_counts)[i]]]) <- c(paste0(names(kinase_raw_counts)[i],"_","R1"),
                                                                       paste0(names(kinase_raw_counts)[i],"_","R2"),
                                                                       paste0(names(kinase_raw_counts)[i],"_","R3"),
                                                                       paste0(names(kinase_raw_counts)[i],"_","R4"),
                                                                       paste0(names(kinase_raw_counts)[i],"_","R5"),
                                                                       paste0(names(kinase_raw_counts)[i],"_","R6"))
    }


# Extract out subset of Reporter intensity
kinase_raw_counts_subset <- list()
for ( i in seq_along(proteinGroups_kinases_ls)){
  kinase_raw_counts_subset[[names(proteinGroups_kinases_ls)[i]]] <-
    proteinGroups_kinases_ls[[i]][,colnames(proteinGroups_kinases_ls[[i]]) %in% c("Reporter.intensity.1","Reporter.intensity.2","Reporter.intensity.3", "Reporter.intensity.4")]
}

# Multiply all values by norm factor and round to the nearest integer
kinase_norm_intensity <- list()
for ( i in seq_along(kinase_raw_counts_subset)){
  kinase_norm_intensity[[names(kinase_raw_counts_subset)[i]]] <-
    round(kinase_raw_counts_subset[[i]]*kinase_norm_per_matrix["norm_factor",i])
}


# Rename the columns
kinase_norm_intensity_rn <- list()
for ( i in seq_along(kinase_norm_intensity)){
  kinase_norm_intensity_rn[[names(kinase_norm_intensity)[i]]] <- kinase_norm_intensity[[i]]
    colnames(kinase_norm_intensity_rn[[names(kinase_norm_intensity)[i]]]) <- c(paste0(names(kinase_norm_intensity)[i],"_","R1"),
                                                                       paste0(names(kinase_norm_intensity)[i],"_","R2"),
                                                                       paste0(names(kinase_norm_intensity)[i],"_","R3"),
                                                                       paste0(names(kinase_norm_intensity)[i],"_","R4"))
    }


# Merge list of dataframe to one big dataframe. We loop through the list with map, create a row names column with rownames_to_column and reduce to a single dataset by doing a full_join by the row names.

kinase_norm_intensity_matrix <- kinase_norm_intensity_rn %>% map( ~ .x %>%
             rownames_to_column('Gene.names'))  %>% 
   reduce(full_join, by = 'Gene.names') 

# Replace all na with 0 value
kinase_norm_intensity_matrix[is.na(kinase_norm_intensity_matrix)] <- "0"

Sample metadata

# Import master sequencing datasheet
TMT_samples_all <-
read.csv(
    "/Data/Input/MIB-MS/Dynamic/240530_MIB_TMT_SampleSheet.csv",
    header = TRUE,
    sep = ",",
    stringsAsFactors = FALSE
  )

# Edit cell line name
TMT_samples_all$Cell.Line <- ifelse(TMT_samples_all$Cell.Line=="PC","CEv3",TMT_samples_all$Cell.Line)

# Convert to factors
TMT_samples_all$replicate <- as.factor(TMT_samples_all$replicate )
TMT_samples_all$Cell.Line <- as.factor(TMT_samples_all$Cell.Line )
TMT_samples_all$Timepoint <- as.factor(TMT_samples_all$Timepoint )
TMT_samples_all$clid <- as.factor(TMT_samples_all$clid)
TMT_samples_all$serum <- as.factor(TMT_samples_all$serum)
TMT_samples_all$condition <- paste0(TMT_samples_all$Cell.Line, "_" ,TMT_samples_all$Timepoint)
TMT_samples_all$condition <- as.factor(TMT_samples_all$condition)
TMT_samples_all$Prep <- as.factor(TMT_samples_all$Prep)
TMT_samples_all$replicate <- as.factor(TMT_samples_all$replicate)
TMT_samples_all$Timepoint <- as.factor(TMT_samples_all$Timepoint)
TMT_samples_all$name <- paste0(TMT_samples_all$condition, "_" ,TMT_samples_all$replicate)
TMT_samples_all$name <- as.factor(TMT_samples_all$name)
TMT_samples_all$Run <- as.factor(TMT_samples_all$Run)
TMT_samples_all$EGFR_TKI_sen <- as.factor(ifelse(TMT_samples_all$Cell.Line %in% c("E4","E5","G1","G5","G8","G12","GR1","GR7"),"Resistant","Sensitive"))


# Check the samplesheet
str(TMT_samples_all)

## 'data.frame':    168 obs. of  18 variables:
##  $ Prep        : Factor w/ 11 levels "Prep_01","Prep_02",..: 1 1 1 1 1 1 1 1 1 1 ...
##  $ Run         : Factor w/ 42 levels "TMT01","TMT02",..: 1 1 1 1 2 2 2 2 3 3 ...
##  $ Number      : int  1 2 3 4 6 7 8 9 11 12 ...
##  $ Sample      : chr  "P0380" "P0381" "P0382" "P0383" ...
##  $ Cell.Line   : Factor w/ 14 levels "C","CE","CEv3",..: 2 2 2 2 1 1 1 1 5 5 ...
##  $ clid        : Factor w/ 14 levels "689","690","691",..: 14 14 14 14 13 13 13 13 12 12 ...
##  $ Timepoint   : Factor w/ 4 levels "0","4","24","48": 1 2 3 4 1 2 3 4 1 2 ...
##  $ serum       : Factor w/ 1 level "starve": 1 1 1 1 1 1 1 1 1 1 ...
##  $ Harvest     : int  180817 180817 180818 180819 180807 180807 180808 180809 180807 180807 ...
##  $ Label_num   : int  126 127 128 129 126 127 128 129 126 127 ...
##  $ Tag         : chr  "R1" "R2" "R3" "R4" ...
##  $ Exp_rep     : int  3 3 3 3 3 3 3 3 3 3 ...
##  $ TMT.Reporter: chr  "TMT01_R1" "TMT01_R2" "TMT01_R3" "TMT01_R4" ...
##  $ label       : chr  "TMT01_R1" "TMT01_R2" "TMT01_R3" "TMT01_R4" ...
##  $ condition   : Factor w/ 56 levels "C_0","C_24","C_4",..: 5 7 6 8 1 3 2 4 17 19 ...
##  $ replicate   : Factor w/ 3 levels "1","2","3": 3 3 3 3 3 3 3 3 3 3 ...
##  $ name        : Factor w/ 168 levels "C_0_1","C_0_2",..: 15 21 18 24 3 9 6 12 51 57 ...
##  $ EGFR_TKI_sen: Factor w/ 2 levels "Resistant","Sensitive": 2 2 2 2 2 2 2 2 2 2 ...

# Make measure column for DEP
TMT_samples_all$measure <- "Intensity"


# Remove cell lines not part of this experiment
TMT_samples_all <- TMT_samples_all[!TMT_samples_all$Cell.Line %in% c("CEv3P", "CP", "CE", "CEv3_MA", "S1","GR1","GR7" ), ]

# Copy over adjusted intensity matrix
se_TMT_all <- kinase_norm_intensity_matrix

se_TMT_all[,2:169]<- as.numeric(unlist(se_TMT_all[,2:169]))

# Remove gene.name
row.names(se_TMT_all) <- se_TMT_all$Gene.names
se_TMT_all <- se_TMT_all[,-which(names(se_TMT_all) %in% c("Gene.names"))]

# Organize for DEP
se_TMT_all$ID <- row.names(se_TMT_all)
se_TMT_all$name <- row.names(se_TMT_all)

# Extract sample names from data matrix
cols <- grep("TMT", colnames(se_TMT_all))

# Check samples match between data matrix and sample sheet
TMT_samples_all$label %in% colnames(se_TMT_all)

##  [1] TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
## [16] TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
## [31] TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
## [46] TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
## [61] TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
## [76] TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE
## [91] TRUE TRUE TRUE TRUE TRUE TRUE

TMT_samples_all$label[!(TMT_samples_all$label %in% colnames(se_TMT_all))]

## character(0)

# Save sample sheet
write.csv(TMT_samples_all, file=paste0(MIB_output,"MIB_TMT_SampleSheet.csv"))

# Save Raw counts
write.csv(se_TMT_all, file=paste0(MIB_output,"MIB_TMT_RawData.csv") )

# Make DEP object
DEP_TMT <- DEP::make_se(se_TMT_all, cols, TMT_samples_all)

DEP_TMT_all

Directories

# Directories for DEP_Bl_Par
dir.create(paste0(MIB_graphs,"DEP_TMT_all"))
dir.create(paste0(MIB_output,"DEP_TMT_all"))
graph_dir <- paste0(MIB_graphs,"DEP_TMT_all/")
output_dir <- paste0(MIB_output,"DEP_TMT_all/")

VST

# Copy over
se_mibs_TMT_raw <- DEP_TMT

# Build summarized experiment object
se_mibs_TMT_vsn <- DEP::normalize_vsn(se_mibs_TMT_raw)

## Warning in vsnSample(v): 34 rows were removed since they contained only NA
## elements.

# Pre and post normalization comparison
DEP::plot_normalization(se_mibs_TMT_raw,se_mibs_TMT_vsn)

Identify missing values

# Plot a heatmap of proteins with missing values
missval_plot <- DEP::plot_missval(se_mibs_TMT_vsn)

missval_plot

# Frequency plot of missing values
freq_plot <- DEP::plot_frequency(se_mibs_TMT_vsn)
freq_plot

# Plot intensity distributions and cumulative fraction of proteins with and without missing values
detect_plot <- DEP::plot_detect(se_mibs_TMT_vsn)

## Warning: Removed 34 rows containing missing values or values outside the scale range
## (`geom_line()`).

detect_plot

## TableGrob (2 x 1) "arrange": 2 grobs
##   z     cells    name           grob
## 1 1 (1-1,1-1) arrange gtable[layout]
## 2 2 (2-2,1-1) arrange gtable[layout]

Impute missing values

# Impute missing data using random draws from a manually defined left-shifted Gaussian distribution (for MNAR)
# scale Numeric(1), Sets the width of the distribution relative to the standard deviation of the original distribution
# shift Numeric(1), Sets the left-shift of the distribution (in standard deviations) from the median of the original distribution.

# # Begin comment out
# 
# # Comment out after running once to lock in imputed values
# set.seed(888)
# se_mibs_TMT_mv <- DEP::impute(se_mibs_TMT_vsn, fun = "man", shift = 3, scale = 0.3)
# 
# # write imputation data to RDS and csv to 'lock in' that specific imputation. Writing to both the impute folder and the output folder simultaneously.
# 
# saveRDS(se_mibs_TMT_mv,file = paste0("/Data/Input/MIB-MS/Dynamic/","se_mibs_TMT_mv",".rds"))
# saveRDS(se_mibs_TMT_mv,file = paste0(MIB_output,"se_mibs_TMT_mv",".rds"))
# 
# write.csv(assays(se_mibs_TMT_mv), paste0("/Data/Input/MIB-MS/Dynamic/","se_mibs_TMT_mv",".csv"))
# write.csv(assays(se_mibs_TMT_mv), paste0(MIB_output,"se_mibs_TMT_mv",".csv"))
# 
# # End comment out

# read in imputation RDS to use for rest of code
se_mibs_TMT_mv <- readRDS(paste0("/Data/Input/MIB-MS/Dynamic/","se_mibs_TMT_mv",".rds"))

# Plot intensity distributions before and after imputation
impute_plot <- plot_imputation(se_mibs_TMT_vsn, se_mibs_TMT_mv)
impute_plot

Batch correction

# Batch correction using Limma
se_mibs_TMT_bc <- se_mibs_TMT_mv
assay(se_mibs_TMT_bc) <- limma::removeBatchEffect(assay(se_mibs_TMT_bc), batch = se_mibs_TMT_bc@colData$Prep , batch2=se_mibs_TMT_bc@colData$Run )

## Coefficients not estimable: batch21 batch22 batch23 batch24 batch26 batch28 batch29 batch210 batch211 batch213 batch214 batch215 batch219 batch221 batch222 batch223 batch224 batch227 batch230 batch231 batch232 batch233 batch234 batch238 batch239 batch240 batch241

## Warning: Partial NA coefficients for 306 probe(s)

QC

Box plots

boxplot(assay(se_mibs_TMT_raw), las=2, ylab="log2 ratio", main="Raw")

boxplot(assay(se_mibs_TMT_vsn), las=2, ylab="log2 ratio", main=paste0("vsn"))

boxplot(assay(se_mibs_TMT_mv), las=2, ylab="log2 ratio", main=paste0("impute missing values"))

boxplot(assay(se_mibs_TMT_bc), las=2, ylab="log2 ratio", main=paste0("batch correction"))

meanSdPlot(se_mibs_TMT_raw)

## Warning: Removed 34 rows containing non-finite outside the scale range
## (`stat_binhex()`).

## Warning: Removed 2 rows containing missing values or values outside the scale range
## (`geom_line()`).

meanSdPlot(se_mibs_TMT_vsn)

## Warning: Removed 34 rows containing non-finite outside the scale range
## (`stat_binhex()`).
## Removed 2 rows containing missing values or values outside the scale range
## (`geom_line()`).

meanSdPlot(se_mibs_TMT_mv)

meanSdPlot(se_mibs_TMT_bc)

PCA plots

PCA cannot be performed with missing values

Imputed values

# PCA with all genes

# Create pca object
pcaData <- PCAtools::pca(assay(se_mibs_TMT_mv), metadata = colData(se_mibs_TMT_mv))

# Save pca data
write.csv(pcaData$rotated, file =paste0(output_dir,"pcaData",".csv"))

# Set colors
color_vector <-
    c(
    'C' = 'black',
    'CEv3' = 'red',
    'E4' = 'deepskyblue4',
    'E5' = 'turquoise3',
    'G1' = 'purple4',
    'G5' = 'mediumorchid',
    'G8' = 'deeppink',
    'G12' = 'pink2'
    )


# plot 5 pricipal components in a pairsplot
pairsplot <- pairsplot(pcaData,
    components = getComponents(pcaData, c(1:5)),
    triangle = TRUE, trianglelabSize = 12,
    hline = 0, vline = 0,
    pointSize = 0.4,
    gridlines.major = FALSE, gridlines.minor = FALSE,
    colby = 'Cell.Line',
    colkey= color_vector,
    title = 'Pairs plot', plotaxes = FALSE,
    legendPosition = 'none',
    margingaps = unit(c(-0.01, -0.01, -0.01, -0.01), 'cm'))

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pairsplot

# save graph as PDF
ggsave(
  filename = paste0(graph_dir,"pca_","pairsplot_mv", ".pdf"),
  plot = pairsplot
  )

## Saving 7 x 5 in image

# PCA 1
p1 <- biplot(pcaData, x = "PC1", y = "PC2",
       lab = NULL ,
       colby = "Cell.Line",
       colkey= color_vector,
       shape = "Timepoint",
       legendPosition = 'right' )

## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.

p1

# save graph as PDF
ggsave(
  filename = paste0(graph_dir,"pca_mv_","cell_line",".pdf"),
  plot = p1
  )

## Saving 7 x 5 in image

Batch corrected values

# PCA with all genes

# Create pca object
pcaData <- PCAtools::pca(assay(se_mibs_TMT_bc), metadata = colData(se_mibs_TMT_bc))

# Save pca data
write.csv(pcaData$rotated, file =paste0(output_dir,"pcaData",".csv"))

# Set colors
color_vector <-
    c(
    'C' = 'black',
    'CEv3' = 'red',
    'E4' = 'deepskyblue4',
    'E5' = 'turquoise3',
    'G1' = 'purple4',
    'G5' = 'mediumorchid',
    'G8' = 'deeppink',
    'G12' = 'pink2'
    )

# plot 5 pricipal components in a pairsplot
pairsplot <- pairsplot(pcaData,
    components = getComponents(pcaData, c(1:5)),
    triangle = TRUE, trianglelabSize = 12,
    hline = 0, vline = 0,
    pointSize = 0.4,
    gridlines.major = FALSE, gridlines.minor = FALSE,
    colby = 'Cell.Line',
    colkey= color_vector,
    title = 'Pairs plot', plotaxes = FALSE,
    legendPosition = 'none',
    margingaps = unit(c(-0.01, -0.01, -0.01, -0.01), 'cm'))

## Scale for colour is already present.
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## Adding another scale for colour, which will replace the existing scale.
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## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.

pairsplot

# save graph as PDF
ggsave(
  filename = paste0(graph_dir,"pca_","pairsplot_bc", ".pdf"),
  plot = pairsplot
  )

## Saving 7 x 5 in image

# PCA 1
p1 <- biplot(pcaData, x = "PC1", y = "PC2",
       lab = NULL ,
       colby = "Cell.Line",
       colkey= color_vector,
       shape = "Timepoint",
       legendPosition = 'right' )

## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.

p1

# save graph as PDF
ggsave(
  filename = paste0(graph_dir,"pca_bc_","cell_line",".pdf"),
  plot = p1
  )

## Saving 7 x 5 in image

DE analysis with limma

Directories

# Directories for MIB
MIB_output

## [1] "/Data/Output/MIB-DYN/"

MIB_graphs

## [1] "/Data/Graphs/MIB-DYN/"

# Directory for full SE obj
dir.create(paste0(MIB_output,"DEP_TMT_all"))

## Warning in dir.create(paste0(MIB_output, "DEP_TMT_all")):
## '/Data/Output/MIB-DYN/DEP_TMT_all' already exists

dir.create(paste0(MIB_graphs,"DEP_TMT_all"))

## Warning in dir.create(paste0(MIB_graphs, "DEP_TMT_all")):
## '/Data/Graphs/MIB-DYN/DEP_TMT_all' already exists

graph_dir <- paste0(MIB_graphs,"DEP_TMT_all/")
output_dir <- paste0(MIB_output,"DEP_TMT_all/")

# Directory for Full SE obj results
dir.create(paste0(output_dir,"Limma_res"))
output_dir_res <- paste0(output_dir,"Limma_res/")
dir.create(paste0(graph_dir,"Limma_res"))
graph_dir_res <- paste0(graph_dir,"Limma_res/")

Comparisons

se_mibs_TMT_mv@colData$condition <- as.factor(se_mibs_TMT_mv@colData$condition)

# comps
comps_factor <- as.vector(factor(paste0(colData(se_mibs_TMT_mv)$condition)))
comps_df <- crossing(comp_num=comps_factor, comp_denom=comps_factor)
comps_df <- subset(comps_df, as.character(comps_df$comp_num) != as.character(comps_df$comp_denom))

# All comps
comps_df_all <- comps_df
comps_df_all <- cbind(comp_type="condition",comps_df_all)
comps_df_all <- cbind(comp_no=1:nrow(comps_df_all),comps_df_all)
comps_df_all$comp_name <- paste(comps_df_all$comp_num, "vs", comps_df_all$comp_denom, sep = "-")
comps_df_all$comp <- paste(comps_df_all$comp_num,comps_df_all$comp_denom, sep="-")
write.csv(comps_df_all, file=paste0(output_dir_res,"comps_all.csv"), col.names = T, row.names = F)

## Warning in write.csv(comps_df_all, file = paste0(output_dir_res,
## "comps_all.csv"), : attempt to set 'col.names' ignored

comps_all_subset <- comps_df_all[,2:4]

# Unique comps only (for only unique combo)
comps_df_unique <- comps_df[!duplicated(t(apply(comps_df, 1, sort))), ]
comps_df_unique <- cbind(comp_type="condition",comps_df_unique )
comps_df_unique <- cbind(comp_no=1:nrow(comps_df_unique),comps_df_unique)
comps_df_unique$comp_name <- paste(comps_df_unique$comp_num, "vs", comps_df_unique$comp_denom, sep = "-")
comps_df_unique$comp <- paste(comps_df_unique$comp_num,comps_df_unique$comp_denom, sep="-")
write.csv(comps_df_unique, file=paste0(output_dir_res,"comps_unique.csv"), col.names = T, row.names = F)

## Warning in write.csv(comps_df_unique, file = paste0(output_dir_res,
## "comps_unique.csv"), : attempt to set 'col.names' ignored

comps_unique_subset <- comps_df_unique[,2:4]

# Comp list to use (all comps)
comps <- comps_df_all

DE Analysis

# Extract gene matrix
gene.matrix <- as.matrix(assay(se_mibs_TMT_mv))

# make design table
cond <- as.vector(se_mibs_TMT_mv@colData$condition)
prep <- as.vector(se_mibs_TMT_mv@colData$Prep)
run <- as.vector(se_mibs_TMT_mv@colData$Run)

# Design formula with prep and run as covariates.
design <- model.matrix(~0 + cond + prep + run)

colnames(design) = gsub("cond","",colnames(design))

head(design)

##   C_0 C_24 C_4 C_48 CEv3_0 CEv3_24 CEv3_4 CEv3_48 E4_0 E4_24 E4_4 E4_48 E5_0
## 1   1    0   0    0      0       0      0       0    0     0    0     0    0
## 2   0    0   1    0      0       0      0       0    0     0    0     0    0
## 3   0    1   0    0      0       0      0       0    0     0    0     0    0
## 4   0    0   0    1      0       0      0       0    0     0    0     0    0
## 5   0    0   0    0      1       0      0       0    0     0    0     0    0
## 6   0    0   0    0      0       0      1       0    0     0    0     0    0
##   E5_24 E5_4 E5_48 G1_0 G1_24 G1_4 G1_48 G12_0 G12_24 G12_4 G12_48 G5_0 G5_24
## 1     0    0     0    0     0    0     0     0      0     0      0    0     0
## 2     0    0     0    0     0    0     0     0      0     0      0    0     0
## 3     0    0     0    0     0    0     0     0      0     0      0    0     0
## 4     0    0     0    0     0    0     0     0      0     0      0    0     0
## 5     0    0     0    0     0    0     0     0      0     0      0    0     0
## 6     0    0     0    0     0    0     0     0      0     0      0    0     0
##   G5_4 G5_48 G8_0 G8_24 G8_4 G8_48 prepPrep_02 prepPrep_03 prepPrep_04
## 1    0     0    0     0    0     0           0           0           0
## 2    0     0    0     0    0     0           0           0           0
## 3    0     0    0     0    0     0           0           0           0
## 4    0     0    0     0    0     0           0           0           0
## 5    0     0    0     0    0     0           1           0           0
## 6    0     0    0     0    0     0           0           1           0
##   prepPrep_05 prepPrep_06 prepPrep_07 prepPrep_08 prepPrep_09 prepPrep_10
## 1           0           0           0           0           0           0
## 2           0           0           0           0           0           0
## 3           0           0           0           0           0           0
## 4           0           0           0           0           0           0
## 5           0           0           0           0           0           0
## 6           0           0           0           0           0           0
##   prepPrep_11 runTMT05 runTMT07 runTMT10 runTMT12 runTMT15 runTMT16 runTMT17
## 1           0        0        0        0        0        0        0        0
## 2           0        0        0        0        0        0        0        0
## 3           0        0        0        0        0        0        0        0
## 4           0        0        0        0        0        0        0        0
## 5           0        1        0        0        0        0        0        0
## 6           0        1        0        0        0        0        0        0
##   runTMT18 runTMT19 runTMT20 runTMT21 runTMT25 runTMT26 runTMT27 runTMT28
## 1        0        0        0        0        0        0        0        0
## 2        0        0        0        0        0        0        0        0
## 3        0        0        0        0        0        0        0        0
## 4        0        0        0        0        0        0        0        0
## 5        0        0        0        0        0        0        0        0
## 6        0        0        0        0        0        0        0        0
##   runTMT29 runTMT30 runTMT34 runTMT35 runTMT36 runTMT37 runTMT38 runTMT39
## 1        0        0        0        0        0        0        0        0
## 2        0        0        0        0        0        0        0        0
## 3        0        0        0        0        0        0        0        0
## 4        0        0        0        0        0        0        0        0
## 5        0        0        0        0        0        0        0        0
## 6        0        0        0        0        0        0        0        0

#limma part analysis
fit1 <- lmFit(gene.matrix, design)

## Coefficients not estimable: prepPrep_11 runTMT10 runTMT12 runTMT15 runTMT19 runTMT21 runTMT25 runTMT27 runTMT29 runTMT30 runTMT34 runTMT35 runTMT36 runTMT37 runTMT38 runTMT39

## Warning: Partial NA coefficients for 306 probe(s)

# Make contrasts
contrast <- makeContrasts(contrasts=comps$comp,levels=design)
fit2 <- eBayes(contrasts.fit(fit1,contrasts = contrast))

# Extract results
mibs_comp_list <- list()
for (i in seq_along(comps$comp)){
  mibs_comp_list[[comps$comp[i]]] <-
    topTreat(fit2, coef=comps$comp[i], number=Inf, adjust.method = "fdr")
  mibs_comp_list[[comps$comp[i]]]$gene <- row.names(mibs_comp_list[[comps$comp[i]]])
}

# Estimate true null
propTrueNull(mibs_comp_list[[1]]$P.Value)

## [1] 0.8237132

# save results
for (i in seq_along(mibs_comp_list)) {
  write.csv(
  mibs_comp_list[[i]],
  file = paste0(output_dir_res,"comp_", i, "_", names(mibs_comp_list)[i], ".csv"),
  row.names = TRUE
  )
}

DE results

# Results
# mibs_comp_list

# Extract results
DEP_TMT_limmaRes <- list()
for (i in seq_along(comps$comp)){
  DEP_TMT_limmaRes[[comps$comp[i]]] <-
    mibs_comp_list[[paste0(comps$comp[i])]]
}

# save results
for (i in seq_along(DEP_TMT_limmaRes)) {
  write.csv(
  DEP_TMT_limmaRes[[i]],
  file = paste0(output_dir_res,"comp_", i, "_", names(DEP_TMT_limmaRes)[i], ".csv"),
  row.names = TRUE
  )
}

DEP_TMT_Res

Directories

# Directories for DEP_TMT_Res
dir.create(paste0(MIB_graphs,"DEP_TMT_Res"))
dir.create(paste0(MIB_output,"DEP_TMT_Res"))
graph_dir <- paste0(MIB_graphs,"DEP_TMT_Res/")
output_dir <- paste0(MIB_output,"DEP_TMT_Res/")

Subset

# Subset 
TMT_samples_all

##        Prep   Run Number Sample Cell.Line clid Timepoint  serum Harvest
## 5   Prep_01 TMT02      6  P0298         C  701         0 starve  180807
## 6   Prep_01 TMT02      7  P0299         C  701         4 starve  180807
## 7   Prep_01 TMT02      8  P0300         C  701        24 starve  180808
## 8   Prep_01 TMT02      9  P0301         C  701        48 starve  180809
## 17  Prep_02 TMT05     21  P0294      CEv3  692         0 starve  180807
## 18  Prep_03 TMT05     22  P0295      CEv3  692         4 starve  180807
## 19  Prep_03 TMT05     23  P0296      CEv3  692        24 starve  180808
## 20  Prep_02 TMT05     24  P0297      CEv3  692        48 starve  180809
## 25  Prep_02 TMT07     31  P0117         C  701         0 starve  180705
## 26  Prep_02 TMT07     32  P0118         C  701         4 starve  180705
## 27  Prep_02 TMT07     33  P0119         C  701        24 starve  180706
## 28  Prep_02 TMT07     34  P0120         C  701        48 starve  180708
## 37  Prep_03 TMT10     46  P0290      CEv3  692         0 starve  180806
## 38  Prep_03 TMT10     47  P0291      CEv3  692         4 starve  180806
## 39  Prep_03 TMT10     48  P0292      CEv3  692        24 starve  180807
## 40  Prep_03 TMT10     49  P0293      CEv3  692        48 starve  180808
## 45  Prep_04 TMT12     56  P0113         C  701         0 starve  180704
## 46  Prep_04 TMT12     57  P0114         C  701         4 starve  180704
## 47  Prep_04 TMT12     58  P0115         C  701        24 starve  180705
## 48  Prep_04 TMT12     59  P0116         C  701        48 starve  180706
## 57  Prep_04 TMT15     71  P0286      CEv3  692         0 starve  180805
## 58  Prep_04 TMT15     72  P0287      CEv3  692         4 starve  180805
## 59  Prep_04 TMT15     73  P0288      CEv3  692        24 starve  180806
## 60  Prep_04 TMT15     74  P0289      CEv3  692        48 starve  180807
## 61  Prep_05 TMT16     76  P0368       G12  698         0 starve  180904
## 62  Prep_05 TMT16     77  P0369       G12  698         4 starve  180904
## 63  Prep_05 TMT16     78  P0370       G12  698        24 starve  180905
## 64  Prep_05 TMT16     79  P0371       G12  698        48 starve  180906
## 65  Prep_05 TMT17     81  P0356        G8  697         0 starve  180826
## 66  Prep_05 TMT17     82  P0357        G8  697         4 starve  180826
## 67  Prep_05 TMT17     83  P0358        G8  697        24 starve  180827
## 68  Prep_05 TMT17     84  P0359        G8  697        48 starve  180828
## 69  Prep_05 TMT18     86  P0344        G5  696         0 starve  180905
## 70  Prep_05 TMT18     87  P0345        G5  696         4 starve  180905
## 71  Prep_05 TMT18     88  P0346        G5  696        24 starve  180906
## 72  Prep_05 TMT18     89  P0347        G5  696        48 starve  180907
## 73  Prep_05 TMT19     91  P0332        G1  695         0 starve  180826
## 74  Prep_05 TMT19     92  P0333        G1  695         4 starve  180826
## 75  Prep_05 TMT19     93  P0334        G1  695        24 starve  180827
## 76  Prep_05 TMT19     94  P0335        G1  695        48 starve  180828
## 77  Prep_06 TMT20     96  P0204        E5  694         0 starve  180724
## 78  Prep_06 TMT20     97  P0205        E5  694         4 starve  180724
## 79  Prep_06 TMT20     98  P0206        E5  694        24 starve  180725
## 80  Prep_06 TMT20     99  P0207        E5  694        48 starve  180726
## 81  Prep_06 TMT21    101  P0189        E4  693         0 starve  180724
## 82  Prep_06 TMT21    102  P0190        E4  693         4 starve  180724
## 83  Prep_06 TMT21    103  P0191        E4  693        24 starve  180724
## 84  Prep_06 TMT21    104  P0192        E4  693        48 starve  180725
## 97  Prep_07 TMT25    121  P0364       G12  698         0 starve  180905
## 98  Prep_07 TMT25    122  P0365       G12  698         4 starve  180905
## 99  Prep_07 TMT25    123  P0366       G12  698        24 starve  180906
## 100 Prep_07 TMT25    124  P0367       G12  698        48 starve  180907
## 101 Prep_07 TMT26    126  P0352        G8  697         0 starve  180825
## 102 Prep_07 TMT26    127  P0353        G8  697         4 starve  180825
## 103 Prep_07 TMT26    128  P0354        G8  697        24 starve  180826
## 104 Prep_07 TMT26    129  P0355        G8  697        48 starve  180827
## 105 Prep_07 TMT27    131  P0340        G5  696         0 starve  180906
## 106 Prep_07 TMT27    132  P0341        G5  696         4 starve  180906
## 107 Prep_07 TMT27    133  P0342        G5  696        24 starve  180907
## 108 Prep_07 TMT27    134  P0343        G5  696        48 starve  180908
## 109 Prep_08 TMT28    136  P0328        G1  695         0 starve  180825
## 110 Prep_08 TMT28    137  P0329        G1  695         4 starve  180825
## 111 Prep_08 TMT28    138  P0330        G1  695        24 starve  180826
## 112 Prep_08 TMT28    139  P0331        G1  695        48 starve  180827
## 113 Prep_08 TMT29    141  P0200        E5  694         0 starve  180723
## 114 Prep_08 TMT29    142  P0201        E5  694         4 starve  180723
## 115 Prep_08 TMT29    143  P0202        E5  694        24 starve  180724
## 116 Prep_08 TMT29    144  P0203        E5  694        48 starve  180725
## 117 Prep_08 TMT30    146  P0185        E4  693         0 starve  180723
## 118 Prep_08 TMT30    147  P0186        E4  693         4 starve  180723
## 119 Prep_08 TMT30    148  P0187        E4  693        24 starve  180724
## 120 Prep_08 TMT30    149  P0188        E4  693        48 starve  180725
## 133 Prep_09 TMT34    166  P0360       G12  698         0 starve  180826
## 134 Prep_09 TMT34    167  P0361       G12  698         4 starve  180826
## 135 Prep_09 TMT34    168  P0362       G12  698        24 starve  180827
## 136 Prep_09 TMT34    169  P0363       G12  698        48 starve  180828
## 137 Prep_10 TMT35    171  P0348        G8  697         0 starve  180824
## 138 Prep_10 TMT35    172  P0349        G8  697         4 starve  180824
## 139 Prep_10 TMT35    173  P0350        G8  697        24 starve  180825
## 140 Prep_10 TMT35    174  P0351        G8  697        48 starve  180826
## 141 Prep_10 TMT36    176  P0336        G5  696         0 starve  180826
## 142 Prep_10 TMT36    177  P0337        G5  696         4 starve  180826
## 143 Prep_10 TMT36    178  P0338        G5  696        24 starve  180827
## 144 Prep_10 TMT36    179  P0339        G5  696        48 starve  180828
## 145 Prep_10 TMT37    181  P0324        G1  695         0 starve  180824
## 146 Prep_10 TMT37    182  P0325        G1  695         4 starve  180824
## 147 Prep_10 TMT37    183  P0326        G1  695        24 starve  180825
## 148 Prep_10 TMT37    184  P0327        G1  695        48 starve  180826
## 149 Prep_10 TMT38    186  P0196        E5  694         0 starve  180722
## 150 Prep_10 TMT38    187  P0197        E5  694         4 starve  180722
## 151 Prep_10 TMT38    188  P0198        E5  694        24 starve  180723
## 152 Prep_10 TMT38    189  P0199        E5  694        48 starve  180724
## 153 Prep_11 TMT39    191  P0181        E4  693         0 starve  180722
## 154 Prep_11 TMT39    192  P0182        E4  693         4 starve  180722
## 155 Prep_11 TMT39    193  P0183        E4  693        24 starve  180723
## 156 Prep_11 TMT39    194  P0184        E4  693        48 starve  180724
##     Label_num Tag Exp_rep TMT.Reporter    label condition replicate      name
## 5         126  R1       3     TMT02_R1 TMT02_R1       C_0         3     C_0_3
## 6         127  R2       3     TMT02_R2 TMT02_R2       C_4         3     C_4_3
## 7         128  R3       3     TMT02_R3 TMT02_R3      C_24         3    C_24_3
## 8         129  R4       3     TMT02_R4 TMT02_R4      C_48         3    C_48_3
## 17        126  R1       3     TMT05_R1 TMT05_R1    CEv3_0         3  CEv3_0_3
## 18        127  R2       3     TMT05_R2 TMT05_R2    CEv3_4         3  CEv3_4_3
## 19        128  R3       3     TMT05_R3 TMT05_R3   CEv3_24         3 CEv3_24_3
## 20        129  R4       3     TMT05_R4 TMT05_R4   CEv3_48         3 CEv3_48_3
## 25        126  R1       2     TMT07_R1 TMT07_R1       C_0         2     C_0_2
## 26        127  R2       2     TMT07_R2 TMT07_R2       C_4         2     C_4_2
## 27        128  R3       2     TMT07_R3 TMT07_R3      C_24         2    C_24_2
## 28        129  R4       2     TMT07_R4 TMT07_R4      C_48         2    C_48_2
## 37        126  R1       2     TMT10_R1 TMT10_R1    CEv3_0         2  CEv3_0_2
## 38        127  R2       2     TMT10_R2 TMT10_R2    CEv3_4         2  CEv3_4_2
## 39        128  R3       2     TMT10_R3 TMT10_R3   CEv3_24         2 CEv3_24_2
## 40        129  R4       2     TMT10_R4 TMT10_R4   CEv3_48         2 CEv3_48_2
## 45        126  R1       1     TMT12_R1 TMT12_R1       C_0         1     C_0_1
## 46        127  R2       1     TMT12_R2 TMT12_R2       C_4         1     C_4_1
## 47        128  R3       1     TMT12_R3 TMT12_R3      C_24         1    C_24_1
## 48        129  R4       1     TMT12_R4 TMT12_R4      C_48         1    C_48_1
## 57        126  R1       1     TMT15_R1 TMT15_R1    CEv3_0         1  CEv3_0_1
## 58        127  R2       1     TMT15_R2 TMT15_R2    CEv3_4         1  CEv3_4_1
## 59        128  R3       1     TMT15_R3 TMT15_R3   CEv3_24         1 CEv3_24_1
## 60        129  R4       1     TMT15_R4 TMT15_R4   CEv3_48         1 CEv3_48_1
## 61        126  R1       3     TMT16_R1 TMT16_R1     G12_0         3   G12_0_3
## 62        127  R2       3     TMT16_R2 TMT16_R2     G12_4         3   G12_4_3
## 63        128  R3       3     TMT16_R3 TMT16_R3    G12_24         3  G12_24_3
## 64        129  R4       3     TMT16_R4 TMT16_R4    G12_48         3  G12_48_3
## 65        126  R1       3     TMT17_R1 TMT17_R1      G8_0         3    G8_0_3
## 66        127  R2       3     TMT17_R2 TMT17_R2      G8_4         3    G8_4_3
## 67        128  R3       3     TMT17_R3 TMT17_R3     G8_24         3   G8_24_3
## 68        129  R4       3     TMT17_R4 TMT17_R4     G8_48         3   G8_48_3
## 69        126  R1       3     TMT18_R1 TMT18_R1      G5_0         3    G5_0_3
## 70        127  R2       3     TMT18_R2 TMT18_R2      G5_4         3    G5_4_3
## 71        128  R3       3     TMT18_R3 TMT18_R3     G5_24         3   G5_24_3
## 72        129  R4       3     TMT18_R4 TMT18_R4     G5_48         3   G5_48_3
## 73        126  R1       3     TMT19_R1 TMT19_R1      G1_0         3    G1_0_3
## 74        127  R2       3     TMT19_R2 TMT19_R2      G1_4         3    G1_4_3
## 75        128  R3       3     TMT19_R3 TMT19_R3     G1_24         3   G1_24_3
## 76        129  R4       3     TMT19_R4 TMT19_R4     G1_48         3   G1_48_3
## 77        126  R1       3     TMT20_R1 TMT20_R1      E5_0         3    E5_0_3
## 78        127  R2       3     TMT20_R2 TMT20_R2      E5_4         3    E5_4_3
## 79        128  R3       3     TMT20_R3 TMT20_R3     E5_24         3   E5_24_3
## 80        129  R4       3     TMT20_R4 TMT20_R4     E5_48         3   E5_48_3
## 81        126  R1       3     TMT21_R1 TMT21_R1      E4_0         3    E4_0_3
## 82        127  R2       3     TMT21_R2 TMT21_R2      E4_4         3    E4_4_3
## 83        128  R3       3     TMT21_R3 TMT21_R3     E4_24         3   E4_24_3
## 84        129  R4       3     TMT21_R4 TMT21_R4     E4_48         3   E4_48_3
## 97        126  R1       2     TMT25_R1 TMT25_R1     G12_0         2   G12_0_2
## 98        127  R2       2     TMT25_R2 TMT25_R2     G12_4         2   G12_4_2
## 99        128  R3       2     TMT25_R3 TMT25_R3    G12_24         2  G12_24_2
## 100       129  R4       2     TMT25_R4 TMT25_R4    G12_48         2  G12_48_2
## 101       126  R1       2     TMT26_R1 TMT26_R1      G8_0         2    G8_0_2
## 102       127  R2       2     TMT26_R2 TMT26_R2      G8_4         2    G8_4_2
## 103       128  R3       2     TMT26_R3 TMT26_R3     G8_24         2   G8_24_2
## 104       129  R4       2     TMT26_R4 TMT26_R4     G8_48         2   G8_48_2
## 105       126  R1       2     TMT27_R1 TMT27_R1      G5_0         2    G5_0_2
## 106       127  R2       2     TMT27_R2 TMT27_R2      G5_4         2    G5_4_2
## 107       128  R3       2     TMT27_R3 TMT27_R3     G5_24         2   G5_24_2
## 108       129  R4       2     TMT27_R4 TMT27_R4     G5_48         2   G5_48_2
## 109       126  R1       2     TMT28_R1 TMT28_R1      G1_0         2    G1_0_2
## 110       127  R2       2     TMT28_R2 TMT28_R2      G1_4         2    G1_4_2
## 111       128  R3       2     TMT28_R3 TMT28_R3     G1_24         2   G1_24_2
## 112       129  R4       2     TMT28_R4 TMT28_R4     G1_48         2   G1_48_2
## 113       126  R1       2     TMT29_R1 TMT29_R1      E5_0         2    E5_0_2
## 114       127  R2       2     TMT29_R2 TMT29_R2      E5_4         2    E5_4_2
## 115       128  R3       2     TMT29_R3 TMT29_R3     E5_24         2   E5_24_2
## 116       129  R4       2     TMT29_R4 TMT29_R4     E5_48         2   E5_48_2
## 117       126  R1       2     TMT30_R1 TMT30_R1      E4_0         2    E4_0_2
## 118       127  R2       2     TMT30_R2 TMT30_R2      E4_4         2    E4_4_2
## 119       128  R3       2     TMT30_R3 TMT30_R3     E4_24         2   E4_24_2
## 120       129  R4       2     TMT30_R4 TMT30_R4     E4_48         2   E4_48_2
## 133       126  R1       1     TMT34_R1 TMT34_R1     G12_0         1   G12_0_1
## 134       127  R2       1     TMT34_R2 TMT34_R2     G12_4         1   G12_4_1
## 135       128  R3       1     TMT34_R3 TMT34_R3    G12_24         1  G12_24_1
## 136       129  R4       1     TMT34_R4 TMT34_R4    G12_48         1  G12_48_1
## 137       126  R1       1     TMT35_R1 TMT35_R1      G8_0         1    G8_0_1
## 138       127  R2       1     TMT35_R2 TMT35_R2      G8_4         1    G8_4_1
## 139       128  R3       1     TMT35_R3 TMT35_R3     G8_24         1   G8_24_1
## 140       129  R4       1     TMT35_R4 TMT35_R4     G8_48         1   G8_48_1
## 141       126  R1       1     TMT36_R1 TMT36_R1      G5_0         1    G5_0_1
## 142       127  R2       1     TMT36_R2 TMT36_R2      G5_4         1    G5_4_1
## 143       128  R3       1     TMT36_R3 TMT36_R3     G5_24         1   G5_24_1
## 144       129  R4       1     TMT36_R4 TMT36_R4     G5_48         1   G5_48_1
## 145       126  R1       1     TMT37_R1 TMT37_R1      G1_0         1    G1_0_1
## 146       127  R2       1     TMT37_R2 TMT37_R2      G1_4         1    G1_4_1
## 147       128  R3       1     TMT37_R3 TMT37_R3     G1_24         1   G1_24_1
## 148       129  R4       1     TMT37_R4 TMT37_R4     G1_48         1   G1_48_1
## 149       126  R1       1     TMT38_R1 TMT38_R1      E5_0         1    E5_0_1
## 150       127  R2       1     TMT38_R2 TMT38_R2      E5_4         1    E5_4_1
## 151       128  R3       1     TMT38_R3 TMT38_R3     E5_24         1   E5_24_1
## 152       129  R4       1     TMT38_R4 TMT38_R4     E5_48         1   E5_48_1
## 153       126  R1       1     TMT39_R1 TMT39_R1      E4_0         1    E4_0_1
## 154       127  R2       1     TMT39_R2 TMT39_R2      E4_4         1    E4_4_1
## 155       128  R3       1     TMT39_R3 TMT39_R3     E4_24         1   E4_24_1
## 156       129  R4       1     TMT39_R4 TMT39_R4     E4_48         1   E4_48_1
##     EGFR_TKI_sen   measure
## 5      Sensitive Intensity
## 6      Sensitive Intensity
## 7      Sensitive Intensity
## 8      Sensitive Intensity
## 17     Sensitive Intensity
## 18     Sensitive Intensity
## 19     Sensitive Intensity
## 20     Sensitive Intensity
## 25     Sensitive Intensity
## 26     Sensitive Intensity
## 27     Sensitive Intensity
## 28     Sensitive Intensity
## 37     Sensitive Intensity
## 38     Sensitive Intensity
## 39     Sensitive Intensity
## 40     Sensitive Intensity
## 45     Sensitive Intensity
## 46     Sensitive Intensity
## 47     Sensitive Intensity
## 48     Sensitive Intensity
## 57     Sensitive Intensity
## 58     Sensitive Intensity
## 59     Sensitive Intensity
## 60     Sensitive Intensity
## 61     Resistant Intensity
## 62     Resistant Intensity
## 63     Resistant Intensity
## 64     Resistant Intensity
## 65     Resistant Intensity
## 66     Resistant Intensity
## 67     Resistant Intensity
## 68     Resistant Intensity
## 69     Resistant Intensity
## 70     Resistant Intensity
## 71     Resistant Intensity
## 72     Resistant Intensity
## 73     Resistant Intensity
## 74     Resistant Intensity
## 75     Resistant Intensity
## 76     Resistant Intensity
## 77     Resistant Intensity
## 78     Resistant Intensity
## 79     Resistant Intensity
## 80     Resistant Intensity
## 81     Resistant Intensity
## 82     Resistant Intensity
## 83     Resistant Intensity
## 84     Resistant Intensity
## 97     Resistant Intensity
## 98     Resistant Intensity
## 99     Resistant Intensity
## 100    Resistant Intensity
## 101    Resistant Intensity
## 102    Resistant Intensity
## 103    Resistant Intensity
## 104    Resistant Intensity
## 105    Resistant Intensity
## 106    Resistant Intensity
## 107    Resistant Intensity
## 108    Resistant Intensity
## 109    Resistant Intensity
## 110    Resistant Intensity
## 111    Resistant Intensity
## 112    Resistant Intensity
## 113    Resistant Intensity
## 114    Resistant Intensity
## 115    Resistant Intensity
## 116    Resistant Intensity
## 117    Resistant Intensity
## 118    Resistant Intensity
## 119    Resistant Intensity
## 120    Resistant Intensity
## 133    Resistant Intensity
## 134    Resistant Intensity
## 135    Resistant Intensity
## 136    Resistant Intensity
## 137    Resistant Intensity
## 138    Resistant Intensity
## 139    Resistant Intensity
## 140    Resistant Intensity
## 141    Resistant Intensity
## 142    Resistant Intensity
## 143    Resistant Intensity
## 144    Resistant Intensity
## 145    Resistant Intensity
## 146    Resistant Intensity
## 147    Resistant Intensity
## 148    Resistant Intensity
## 149    Resistant Intensity
## 150    Resistant Intensity
## 151    Resistant Intensity
## 152    Resistant Intensity
## 153    Resistant Intensity
## 154    Resistant Intensity
## 155    Resistant Intensity
## 156    Resistant Intensity

TMT_samples_Res <- subset(TMT_samples_all,TMT_samples_all$Cell.Line %in% c("CEv3","E4","E5","G1","G5","G8","G12"))

# Subset DEP object
DEP_TMT_Res <- se_mibs_TMT_bc[,paste0(TMT_samples_Res$name)]

# Save sample sheet
write.csv(TMT_samples_Res, file=paste0(output_dir,"DEP_TMT_Res",".csv"))

# Save DEP_2019_parental as RDS
saveRDS(DEP_TMT_Res, file = paste0(output_dir,format(Sys.Date(),"%y%m%d"),"_","DEP_TMT_Res",".rds"))

PCA

# PCA with all genes

# Create pca object
pcaData <- PCAtools::pca(assay(DEP_TMT_Res), metadata = colData(DEP_TMT_Res))

# Save pca data
write.csv(pcaData$rotated, file =paste0(output_dir,"pcaData",".csv"))

# Set colors
color_vector <-
    c(
    'C' = 'black',
    'CEv3' = 'red',
    'E4' = 'deepskyblue4',
    'E5' = 'turquoise3',
    'G1' = 'purple4',
    'G5' = 'mediumorchid',
    'G8' = 'deeppink',
    'G12' = 'pink2'
    )

# plot 5 pricipal components in a pairsplot
pairsplot <- pairsplot(pcaData,
    components = getComponents(pcaData, c(1:5)),
    triangle = TRUE, trianglelabSize = 12,
    hline = 0, vline = 0,
    pointSize = 0.4,
    gridlines.major = FALSE, gridlines.minor = FALSE,
    colby = 'Cell.Line',
    colkey= color_vector,
    title = 'Pairs plot', plotaxes = FALSE,
    legendPosition = 'none',
    margingaps = unit(c(-0.01, -0.01, -0.01, -0.01), 'cm'))

## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.

pairsplot

# save graph as PDF
ggsave(
  filename = paste0(graph_dir,"pca_","pairsplot", ".pdf"),
  plot = pairsplot
  )

## Saving 7 x 5 in image

# PCA 1
p1 <- biplot(pcaData, x = "PC1", y = "PC2",
       lab = NULL ,
       colby = "Cell.Line",
       colkey= color_vector,
       shape = "Timepoint",
       legendPosition = 'right' )

## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.

p1

# save graph as PDF
ggsave(
  filename = paste0(graph_dir,"pca_vsd_500_","cell_line",".pdf"),
  plot = p1
  )

## Saving 7 x 5 in image

Euclidian distances

# calculate sample euclidian distances
sampleDists <- dist(t(assay(DEP_TMT_Res)))

# plot sample euclidian distances via a heatmap
sampleDistMatrix <- as.matrix( sampleDists )

# Complex Heatmap annotation labels
annotation_col<-
  HeatmapAnnotation(df = data.frame(Model = DEP_TMT_Res@colData$Cell.Line,
                                    Time = DEP_TMT_Res@colData$Timepoint),
  col = list(Model =
  c(
    'C' = 'black',
    'CEv3' = 'red',
    'E4' = 'deepskyblue4',
    'E5' = 'turquoise3',
    'G1' = 'purple4',
    'G5' = 'mediumorchid',
    'G8' = 'deeppink',
    'G12' = 'pink2'
  ),
  Time =
  c(
    '0' = 'black',
    '4' = 'purple',
    '24' = 'darkgreen',
    '48' = 'darkorange'
  ))
  )

# Heatmap of euclidian distances
ComplexHeatmap::Heatmap(
      sampleDistMatrix,
      name = " ",
      column_title = "Eculidian Distances",
      show_column_names = FALSE,
      show_row_names = FALSE,
      clustering_distance_rows = "euclidean",
      clustering_distance_columns = "euclidean",
      clustering_method_rows = "complete",
      clustering_method_columns = "complete",
      col = colorRampPalette( rev(brewer.pal(9, "Blues")) )(255),
      top_annotation = annotation_col
    )

TVG (labeled)

# DEP_TMT_Res

# Directories 
dir.create(paste0(output_dir,"Heatmap_TVG"))
output_dir_Heatmap_TVG <- paste0(output_dir,"Heatmap_TVG/")
dir.create(paste0(graph_dir,"Heatmap_TVG"))
graph_dir_Heatmap_TVG <- paste0(graph_dir,"Heatmap_TVG/")

# pHeatmap of Top X DEG on transformed data
tvg <- c(100,50,25,10)
topVarGenesX_list <- list()

# pHeatmap of Top X DEG on transformed data

# Extract out distances
mat_list <- list()
for (i in seq_along(tvg)) {
  topVarGenesX_list[[paste0(tvg[i])]] <- head( order( rowVars( as.matrix(assay(DEP_TMT_Res)) ), decreasing=TRUE ), tvg[[i]] )
}
for (i in seq_along(tvg)) {
  mat_list[[paste0(tvg[i])]] <- assay(DEP_TMT_Res)[ topVarGenesX_list[[i]], ]
}

# Heatmap annotation labels
annotation_col<-
  HeatmapAnnotation(df = data.frame(Model = DEP_TMT_Res@colData$Cell.Line,
                                    Time = DEP_TMT_Res@colData$Timepoint),
  col = list(Model =
  c(
    'C' = 'black',
    'CEv3' = 'red',
    'E4' = 'deepskyblue4',
    'E5' = 'turquoise3',
    'G1' = 'purple4',
    'G5' = 'mediumorchid',
    'G8' = 'deeppink',
    'G12' = 'pink2'
  ),
  Time =
  c(
    '0' = 'black',
    '4' = 'purple',
    '24' = 'darkgreen',
    '48' = 'darkorange'
  ))
  )

# Generate Unlabeled Heatmap
p_list <- list()
for (i in seq_along(mat_list)) {
p_list[[paste0(names(mat_list)[[i]])]] <- ComplexHeatmap::Heatmap(
  t(scale(t(mat_list[[i]]))),
  top_annotation = annotation_col,
  name = " ",
  column_title = paste0(names(mat_list)[i],"_TVG"),
  show_column_names = F,
  show_row_names = F
  )
print(p_list[[i]])
}

# Save Heatmap
for (i in seq_along(p_list)) {
pdf(file=paste0(graph_dir_Heatmap_TVG, "CHeatmap_"  , names(p_list)[[i]], ".pdf"))
draw(p_list[[i]])
dev.off()
}


# Generate labeled Heatmap
p_list <- list()
for (i in seq_along(mat_list)) {
p_list[[paste0(names(mat_list)[[i]])]] <- ComplexHeatmap::Heatmap(
  t(scale(t(mat_list[[i]]))),
  #column_order=row.names(DEP_TMT_Res@colData[order(DEP_TMT_Res@colData$clid),]),
  top_annotation = annotation_col,
  column_split = factor(DEP_TMT_Res@colData$clid),
  name = " ",
  column_title = paste0(names(mat_list)[i],"_TVG"),
  show_column_names = F,
  show_row_names = T
  )
print(p_list[[i]])
}

# Save Heatmap
for (i in seq_along(p_list)) {
pdf(file=paste0(graph_dir_Heatmap_TVG, "CHeatmapLabel_"  , names(p_list)[[i]], ".pdf"))
draw(p_list[[i]])
dev.off()
}

# Graphics termination 
if(!is.null(dev.list())) dev.off()

## null device 
##           1

if(!is.null(dev.list())) graphics.off()

Limma

Comparisons

# Directories 
dir.create(paste0(output_dir,"Limma_res"))
output_dir_res <- paste0(output_dir,"Limma_res/")
dir.create(paste0(graph_dir,"Limma_res"))
graph_dir_res <- paste0(graph_dir,"Limma_res/")

# comps
comps_factor <- as.vector(factor(paste0(colData(DEP_TMT_Res)$condition)))
comps_df <- crossing(comp_num=comps_factor, comp_denom=comps_factor)
comps_df <- subset(comps_df, as.character(comps_df$comp_num) != as.character(comps_df$comp_denom))

# All comps
comps_df_all <- comps_df
comps_df_all <- cbind(comp_type="condition",comps_df_all)
comps_df_all <- cbind(comp_no=1:nrow(comps_df_all),comps_df_all)
comps_df_all$comp_name <- paste(comps_df_all$comp_num, "vs", comps_df_all$comp_denom, sep = "-")
comps_df_all$comp <- paste(comps_df_all$comp_num,comps_df_all$comp_denom, sep="-")
write.csv(comps_df_all, file=paste0(output_dir_res,"comps_all.csv"), col.names = T, row.names = F)

## Warning in write.csv(comps_df_all, file = paste0(output_dir_res,
## "comps_all.csv"), : attempt to set 'col.names' ignored

comps_all_subset <- comps_df_all[,2:4]

# Unique comps only (for only unique combo)
comps_df_unique <- comps_df[!duplicated(t(apply(comps_df, 1, sort))), ]
comps_df_unique <- cbind(comp_type="condition",comps_df_unique )
comps_df_unique <- cbind(comp_no=1:nrow(comps_df_unique),comps_df_unique)
comps_df_unique$comp_name <- paste(comps_df_unique$comp_num, "vs", comps_df_unique$comp_denom, sep = "-")
comps_df_unique$comp <- paste(comps_df_unique$comp_num,comps_df_unique$comp_denom, sep="-")
write.csv(comps_df_unique, file=paste0(output_dir_res,"comps_unique.csv"), col.names = T, row.names = F)

## Warning in write.csv(comps_df_unique, file = paste0(output_dir_res,
## "comps_unique.csv"), : attempt to set 'col.names' ignored

comps_unique_subset <- comps_df_unique[,2:4]

# Comp list to use (all comps)
comps <- comps_df_all

DE results

# Extract
# Results
# mibs_comp_list

# Directories 
dir.create(paste0(output_dir,"Limma_res"))

## Warning in dir.create(paste0(output_dir, "Limma_res")):
## '/Data/Output/MIB-DYN/DEP_TMT_Res/Limma_res' already exists

output_dir_res <- paste0(output_dir,"Limma_res/")
dir.create(paste0(graph_dir,"Limma_res"))

## Warning in dir.create(paste0(graph_dir, "Limma_res")):
## '/Data/Graphs/MIB-DYN/DEP_TMT_Res/Limma_res' already exists

graph_dir_res <- paste0(graph_dir,"Limma_res/")

# Extract results
DEP_TMT_limmaRes <- list()
for (i in seq_along(comps$comp)){
  DEP_TMT_limmaRes[[comps$comp[i]]] <-
    mibs_comp_list[[paste0(comps$comp[i])]]
}

# save results
for (i in seq_along(DEP_TMT_limmaRes)) {
  write.csv(
  DEP_TMT_limmaRes[[i]],
  file = paste0(output_dir_res,"comp_", i, "_", names(DEP_TMT_limmaRes)[i], ".csv"),
  row.names = TRUE
  )
}

Summarize Results

# Directories 
dir.create(paste0(output_dir,"Limma_res"))

## Warning in dir.create(paste0(output_dir, "Limma_res")):
## '/Data/Output/MIB-DYN/DEP_TMT_Res/Limma_res' already exists

output_dir_res <- paste0(output_dir,"Limma_res/")
dir.create(paste0(graph_dir,"Limma_res"))

## Warning in dir.create(paste0(graph_dir, "Limma_res")):
## '/Data/Graphs/MIB-DYN/DEP_TMT_Res/Limma_res' already exists

graph_dir_res <- paste0(graph_dir,"Limma_res/")

# Extract comps of interest
coi <- comps_df_all[comps_df_all$comp_denom=="CEv3_0",]
comp_list_oi <- DEP_TMT_limmaRes[names(DEP_TMT_limmaRes) %in% coi$comp]
comp_list_oi <- lapply(comp_list_oi,data.frame)

# Label list of DF
for (i in seq_along(comp_list_oi)){
  colnames(comp_list_oi[[i]]) <- paste0(colnames(comp_list_oi[[i]]),"_",names(comp_list_oi)[[i]])
  comp_list_oi[[i]]$Gene_ID <- row.names(comp_list_oi[[i]])
}

# Left_join comps of interest to 1x spreadsheet
comp_list_oi_master <- Reduce(function(x, y) left_join(x, y, by="Gene_ID"), comp_list_oi)
row.names(comp_list_oi_master) <- comp_list_oi_master$Gene_ID
comp_list_oi_master <- comp_list_oi_master[,-which(colnames(comp_list_oi_master)=="Gene_ID")]
comp_list_oi_master <- comp_list_oi_master[,-which(colnames(comp_list_oi_master)%in% paste0(grep("gene_",colnames(comp_list_oi_master), value = T)))]

# Subset to spreadsheets
comp_list_oi_padj <- comp_list_oi_master[,grep("adj.P.Val_", colnames(comp_list_oi_master))]
comp_list_oi_pvalue <- comp_list_oi_master[,grep("P.Value_", colnames(comp_list_oi_master))]
comp_list_oi_l2fc <- comp_list_oi_master[,grep("logFC_", colnames(comp_list_oi_master))]

# Save spreadsheets
write.csv(comp_list_oi_padj, file = paste0(output_dir_res,"All_coi_","padj",".csv"))
write.csv(comp_list_oi_pvalue, file = paste0(output_dir_res,"All_coi_","pvalue",".csv"))
write.csv(comp_list_oi_l2fc, file = paste0(output_dir_res,"All_coi_","l2fc",".csv"))

Volcano Plots

# Directory for volcano plots
dir.create(paste0(graph_dir,"/","volcano"))
graph_dir_vol <- paste0(graph_dir,"volcano/")

# Plot Volcano
volcano_plots_ls <- list()
for (i in seq_along(DEP_TMT_limmaRes)){
  plot(volcano_plots_ls[[names(DEP_TMT_limmaRes)[i]]] <- 
    EnhancedVolcano(
      DEP_TMT_limmaRes[[i]], 
      lab=row.names(DEP_TMT_limmaRes[[i]]),
      x="logFC",
      y="adj.P.Val",
      title= paste0("MIBs ",names(DEP_TMT_limmaRes)[[i]]),
      pCutoff =qval, 
      FCcutoff =lfc,
      pointSize = 2.0, 
      labSize = 3.0 
    ))}

# Save Volcano
for (i in seq_along(volcano_plots_ls)){
ggsave(filename = paste0(graph_dir_vol,"comp_", i, "_", names(volcano_plots_ls)[[i]],".pdf"), plot=volcano_plots_ls[[i]])
}

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## Saving 7 x 5 in image
## Saving 7 x 5 in image
## Saving 7 x 5 in image
## Saving 7 x 5 in image
## Saving 7 x 5 in image
## Saving 7 x 5 in image
## Saving 7 x 5 in image
## Saving 7 x 5 in image
## Saving 7 x 5 in image
## Saving 7 x 5 in image
## Saving 7 x 5 in image
## Saving 7 x 5 in image
## Saving 7 x 5 in image
## Saving 7 x 5 in image
## Saving 7 x 5 in image
## Saving 7 x 5 in image
## Saving 7 x 5 in image
## Saving 7 x 5 in image
## Saving 7 x 5 in image
## Saving 7 x 5 in image
## Saving 7 x 5 in image
## Saving 7 x 5 in image
## Saving 7 x 5 in image

# Graphics termination 
if(!is.null(dev.list())) dev.off()

## null device 
##           1

if(!is.null(dev.list())) graphics.off()

DEP_TMT_Par

Directories

# Directories for DEP_TMT_Par
dir.create(paste0(MIB_graphs,"DEP_TMT_Par"))
dir.create(paste0(MIB_output,"DEP_TMT_Par"))
graph_dir <- paste0(MIB_graphs,"DEP_TMT_Par/")
output_dir <- paste0(MIB_output,"DEP_TMT_Par/")

Subset

# Subset 
TMT_samples_all

##        Prep   Run Number Sample Cell.Line clid Timepoint  serum Harvest
## 5   Prep_01 TMT02      6  P0298         C  701         0 starve  180807
## 6   Prep_01 TMT02      7  P0299         C  701         4 starve  180807
## 7   Prep_01 TMT02      8  P0300         C  701        24 starve  180808
## 8   Prep_01 TMT02      9  P0301         C  701        48 starve  180809
## 17  Prep_02 TMT05     21  P0294      CEv3  692         0 starve  180807
## 18  Prep_03 TMT05     22  P0295      CEv3  692         4 starve  180807
## 19  Prep_03 TMT05     23  P0296      CEv3  692        24 starve  180808
## 20  Prep_02 TMT05     24  P0297      CEv3  692        48 starve  180809
## 25  Prep_02 TMT07     31  P0117         C  701         0 starve  180705
## 26  Prep_02 TMT07     32  P0118         C  701         4 starve  180705
## 27  Prep_02 TMT07     33  P0119         C  701        24 starve  180706
## 28  Prep_02 TMT07     34  P0120         C  701        48 starve  180708
## 37  Prep_03 TMT10     46  P0290      CEv3  692         0 starve  180806
## 38  Prep_03 TMT10     47  P0291      CEv3  692         4 starve  180806
## 39  Prep_03 TMT10     48  P0292      CEv3  692        24 starve  180807
## 40  Prep_03 TMT10     49  P0293      CEv3  692        48 starve  180808
## 45  Prep_04 TMT12     56  P0113         C  701         0 starve  180704
## 46  Prep_04 TMT12     57  P0114         C  701         4 starve  180704
## 47  Prep_04 TMT12     58  P0115         C  701        24 starve  180705
## 48  Prep_04 TMT12     59  P0116         C  701        48 starve  180706
## 57  Prep_04 TMT15     71  P0286      CEv3  692         0 starve  180805
## 58  Prep_04 TMT15     72  P0287      CEv3  692         4 starve  180805
## 59  Prep_04 TMT15     73  P0288      CEv3  692        24 starve  180806
## 60  Prep_04 TMT15     74  P0289      CEv3  692        48 starve  180807
## 61  Prep_05 TMT16     76  P0368       G12  698         0 starve  180904
## 62  Prep_05 TMT16     77  P0369       G12  698         4 starve  180904
## 63  Prep_05 TMT16     78  P0370       G12  698        24 starve  180905
## 64  Prep_05 TMT16     79  P0371       G12  698        48 starve  180906
## 65  Prep_05 TMT17     81  P0356        G8  697         0 starve  180826
## 66  Prep_05 TMT17     82  P0357        G8  697         4 starve  180826
## 67  Prep_05 TMT17     83  P0358        G8  697        24 starve  180827
## 68  Prep_05 TMT17     84  P0359        G8  697        48 starve  180828
## 69  Prep_05 TMT18     86  P0344        G5  696         0 starve  180905
## 70  Prep_05 TMT18     87  P0345        G5  696         4 starve  180905
## 71  Prep_05 TMT18     88  P0346        G5  696        24 starve  180906
## 72  Prep_05 TMT18     89  P0347        G5  696        48 starve  180907
## 73  Prep_05 TMT19     91  P0332        G1  695         0 starve  180826
## 74  Prep_05 TMT19     92  P0333        G1  695         4 starve  180826
## 75  Prep_05 TMT19     93  P0334        G1  695        24 starve  180827
## 76  Prep_05 TMT19     94  P0335        G1  695        48 starve  180828
## 77  Prep_06 TMT20     96  P0204        E5  694         0 starve  180724
## 78  Prep_06 TMT20     97  P0205        E5  694         4 starve  180724
## 79  Prep_06 TMT20     98  P0206        E5  694        24 starve  180725
## 80  Prep_06 TMT20     99  P0207        E5  694        48 starve  180726
## 81  Prep_06 TMT21    101  P0189        E4  693         0 starve  180724
## 82  Prep_06 TMT21    102  P0190        E4  693         4 starve  180724
## 83  Prep_06 TMT21    103  P0191        E4  693        24 starve  180724
## 84  Prep_06 TMT21    104  P0192        E4  693        48 starve  180725
## 97  Prep_07 TMT25    121  P0364       G12  698         0 starve  180905
## 98  Prep_07 TMT25    122  P0365       G12  698         4 starve  180905
## 99  Prep_07 TMT25    123  P0366       G12  698        24 starve  180906
## 100 Prep_07 TMT25    124  P0367       G12  698        48 starve  180907
## 101 Prep_07 TMT26    126  P0352        G8  697         0 starve  180825
## 102 Prep_07 TMT26    127  P0353        G8  697         4 starve  180825
## 103 Prep_07 TMT26    128  P0354        G8  697        24 starve  180826
## 104 Prep_07 TMT26    129  P0355        G8  697        48 starve  180827
## 105 Prep_07 TMT27    131  P0340        G5  696         0 starve  180906
## 106 Prep_07 TMT27    132  P0341        G5  696         4 starve  180906
## 107 Prep_07 TMT27    133  P0342        G5  696        24 starve  180907
## 108 Prep_07 TMT27    134  P0343        G5  696        48 starve  180908
## 109 Prep_08 TMT28    136  P0328        G1  695         0 starve  180825
## 110 Prep_08 TMT28    137  P0329        G1  695         4 starve  180825
## 111 Prep_08 TMT28    138  P0330        G1  695        24 starve  180826
## 112 Prep_08 TMT28    139  P0331        G1  695        48 starve  180827
## 113 Prep_08 TMT29    141  P0200        E5  694         0 starve  180723
## 114 Prep_08 TMT29    142  P0201        E5  694         4 starve  180723
## 115 Prep_08 TMT29    143  P0202        E5  694        24 starve  180724
## 116 Prep_08 TMT29    144  P0203        E5  694        48 starve  180725
## 117 Prep_08 TMT30    146  P0185        E4  693         0 starve  180723
## 118 Prep_08 TMT30    147  P0186        E4  693         4 starve  180723
## 119 Prep_08 TMT30    148  P0187        E4  693        24 starve  180724
## 120 Prep_08 TMT30    149  P0188        E4  693        48 starve  180725
## 133 Prep_09 TMT34    166  P0360       G12  698         0 starve  180826
## 134 Prep_09 TMT34    167  P0361       G12  698         4 starve  180826
## 135 Prep_09 TMT34    168  P0362       G12  698        24 starve  180827
## 136 Prep_09 TMT34    169  P0363       G12  698        48 starve  180828
## 137 Prep_10 TMT35    171  P0348        G8  697         0 starve  180824
## 138 Prep_10 TMT35    172  P0349        G8  697         4 starve  180824
## 139 Prep_10 TMT35    173  P0350        G8  697        24 starve  180825
## 140 Prep_10 TMT35    174  P0351        G8  697        48 starve  180826
## 141 Prep_10 TMT36    176  P0336        G5  696         0 starve  180826
## 142 Prep_10 TMT36    177  P0337        G5  696         4 starve  180826
## 143 Prep_10 TMT36    178  P0338        G5  696        24 starve  180827
## 144 Prep_10 TMT36    179  P0339        G5  696        48 starve  180828
## 145 Prep_10 TMT37    181  P0324        G1  695         0 starve  180824
## 146 Prep_10 TMT37    182  P0325        G1  695         4 starve  180824
## 147 Prep_10 TMT37    183  P0326        G1  695        24 starve  180825
## 148 Prep_10 TMT37    184  P0327        G1  695        48 starve  180826
## 149 Prep_10 TMT38    186  P0196        E5  694         0 starve  180722
## 150 Prep_10 TMT38    187  P0197        E5  694         4 starve  180722
## 151 Prep_10 TMT38    188  P0198        E5  694        24 starve  180723
## 152 Prep_10 TMT38    189  P0199        E5  694        48 starve  180724
## 153 Prep_11 TMT39    191  P0181        E4  693         0 starve  180722
## 154 Prep_11 TMT39    192  P0182        E4  693         4 starve  180722
## 155 Prep_11 TMT39    193  P0183        E4  693        24 starve  180723
## 156 Prep_11 TMT39    194  P0184        E4  693        48 starve  180724
##     Label_num Tag Exp_rep TMT.Reporter    label condition replicate      name
## 5         126  R1       3     TMT02_R1 TMT02_R1       C_0         3     C_0_3
## 6         127  R2       3     TMT02_R2 TMT02_R2       C_4         3     C_4_3
## 7         128  R3       3     TMT02_R3 TMT02_R3      C_24         3    C_24_3
## 8         129  R4       3     TMT02_R4 TMT02_R4      C_48         3    C_48_3
## 17        126  R1       3     TMT05_R1 TMT05_R1    CEv3_0         3  CEv3_0_3
## 18        127  R2       3     TMT05_R2 TMT05_R2    CEv3_4         3  CEv3_4_3
## 19        128  R3       3     TMT05_R3 TMT05_R3   CEv3_24         3 CEv3_24_3
## 20        129  R4       3     TMT05_R4 TMT05_R4   CEv3_48         3 CEv3_48_3
## 25        126  R1       2     TMT07_R1 TMT07_R1       C_0         2     C_0_2
## 26        127  R2       2     TMT07_R2 TMT07_R2       C_4         2     C_4_2
## 27        128  R3       2     TMT07_R3 TMT07_R3      C_24         2    C_24_2
## 28        129  R4       2     TMT07_R4 TMT07_R4      C_48         2    C_48_2
## 37        126  R1       2     TMT10_R1 TMT10_R1    CEv3_0         2  CEv3_0_2
## 38        127  R2       2     TMT10_R2 TMT10_R2    CEv3_4         2  CEv3_4_2
## 39        128  R3       2     TMT10_R3 TMT10_R3   CEv3_24         2 CEv3_24_2
## 40        129  R4       2     TMT10_R4 TMT10_R4   CEv3_48         2 CEv3_48_2
## 45        126  R1       1     TMT12_R1 TMT12_R1       C_0         1     C_0_1
## 46        127  R2       1     TMT12_R2 TMT12_R2       C_4         1     C_4_1
## 47        128  R3       1     TMT12_R3 TMT12_R3      C_24         1    C_24_1
## 48        129  R4       1     TMT12_R4 TMT12_R4      C_48         1    C_48_1
## 57        126  R1       1     TMT15_R1 TMT15_R1    CEv3_0         1  CEv3_0_1
## 58        127  R2       1     TMT15_R2 TMT15_R2    CEv3_4         1  CEv3_4_1
## 59        128  R3       1     TMT15_R3 TMT15_R3   CEv3_24         1 CEv3_24_1
## 60        129  R4       1     TMT15_R4 TMT15_R4   CEv3_48         1 CEv3_48_1
## 61        126  R1       3     TMT16_R1 TMT16_R1     G12_0         3   G12_0_3
## 62        127  R2       3     TMT16_R2 TMT16_R2     G12_4         3   G12_4_3
## 63        128  R3       3     TMT16_R3 TMT16_R3    G12_24         3  G12_24_3
## 64        129  R4       3     TMT16_R4 TMT16_R4    G12_48         3  G12_48_3
## 65        126  R1       3     TMT17_R1 TMT17_R1      G8_0         3    G8_0_3
## 66        127  R2       3     TMT17_R2 TMT17_R2      G8_4         3    G8_4_3
## 67        128  R3       3     TMT17_R3 TMT17_R3     G8_24         3   G8_24_3
## 68        129  R4       3     TMT17_R4 TMT17_R4     G8_48         3   G8_48_3
## 69        126  R1       3     TMT18_R1 TMT18_R1      G5_0         3    G5_0_3
## 70        127  R2       3     TMT18_R2 TMT18_R2      G5_4         3    G5_4_3
## 71        128  R3       3     TMT18_R3 TMT18_R3     G5_24         3   G5_24_3
## 72        129  R4       3     TMT18_R4 TMT18_R4     G5_48         3   G5_48_3
## 73        126  R1       3     TMT19_R1 TMT19_R1      G1_0         3    G1_0_3
## 74        127  R2       3     TMT19_R2 TMT19_R2      G1_4         3    G1_4_3
## 75        128  R3       3     TMT19_R3 TMT19_R3     G1_24         3   G1_24_3
## 76        129  R4       3     TMT19_R4 TMT19_R4     G1_48         3   G1_48_3
## 77        126  R1       3     TMT20_R1 TMT20_R1      E5_0         3    E5_0_3
## 78        127  R2       3     TMT20_R2 TMT20_R2      E5_4         3    E5_4_3
## 79        128  R3       3     TMT20_R3 TMT20_R3     E5_24         3   E5_24_3
## 80        129  R4       3     TMT20_R4 TMT20_R4     E5_48         3   E5_48_3
## 81        126  R1       3     TMT21_R1 TMT21_R1      E4_0         3    E4_0_3
## 82        127  R2       3     TMT21_R2 TMT21_R2      E4_4         3    E4_4_3
## 83        128  R3       3     TMT21_R3 TMT21_R3     E4_24         3   E4_24_3
## 84        129  R4       3     TMT21_R4 TMT21_R4     E4_48         3   E4_48_3
## 97        126  R1       2     TMT25_R1 TMT25_R1     G12_0         2   G12_0_2
## 98        127  R2       2     TMT25_R2 TMT25_R2     G12_4         2   G12_4_2
## 99        128  R3       2     TMT25_R3 TMT25_R3    G12_24         2  G12_24_2
## 100       129  R4       2     TMT25_R4 TMT25_R4    G12_48         2  G12_48_2
## 101       126  R1       2     TMT26_R1 TMT26_R1      G8_0         2    G8_0_2
## 102       127  R2       2     TMT26_R2 TMT26_R2      G8_4         2    G8_4_2
## 103       128  R3       2     TMT26_R3 TMT26_R3     G8_24         2   G8_24_2
## 104       129  R4       2     TMT26_R4 TMT26_R4     G8_48         2   G8_48_2
## 105       126  R1       2     TMT27_R1 TMT27_R1      G5_0         2    G5_0_2
## 106       127  R2       2     TMT27_R2 TMT27_R2      G5_4         2    G5_4_2
## 107       128  R3       2     TMT27_R3 TMT27_R3     G5_24         2   G5_24_2
## 108       129  R4       2     TMT27_R4 TMT27_R4     G5_48         2   G5_48_2
## 109       126  R1       2     TMT28_R1 TMT28_R1      G1_0         2    G1_0_2
## 110       127  R2       2     TMT28_R2 TMT28_R2      G1_4         2    G1_4_2
## 111       128  R3       2     TMT28_R3 TMT28_R3     G1_24         2   G1_24_2
## 112       129  R4       2     TMT28_R4 TMT28_R4     G1_48         2   G1_48_2
## 113       126  R1       2     TMT29_R1 TMT29_R1      E5_0         2    E5_0_2
## 114       127  R2       2     TMT29_R2 TMT29_R2      E5_4         2    E5_4_2
## 115       128  R3       2     TMT29_R3 TMT29_R3     E5_24         2   E5_24_2
## 116       129  R4       2     TMT29_R4 TMT29_R4     E5_48         2   E5_48_2
## 117       126  R1       2     TMT30_R1 TMT30_R1      E4_0         2    E4_0_2
## 118       127  R2       2     TMT30_R2 TMT30_R2      E4_4         2    E4_4_2
## 119       128  R3       2     TMT30_R3 TMT30_R3     E4_24         2   E4_24_2
## 120       129  R4       2     TMT30_R4 TMT30_R4     E4_48         2   E4_48_2
## 133       126  R1       1     TMT34_R1 TMT34_R1     G12_0         1   G12_0_1
## 134       127  R2       1     TMT34_R2 TMT34_R2     G12_4         1   G12_4_1
## 135       128  R3       1     TMT34_R3 TMT34_R3    G12_24         1  G12_24_1
## 136       129  R4       1     TMT34_R4 TMT34_R4    G12_48         1  G12_48_1
## 137       126  R1       1     TMT35_R1 TMT35_R1      G8_0         1    G8_0_1
## 138       127  R2       1     TMT35_R2 TMT35_R2      G8_4         1    G8_4_1
## 139       128  R3       1     TMT35_R3 TMT35_R3     G8_24         1   G8_24_1
## 140       129  R4       1     TMT35_R4 TMT35_R4     G8_48         1   G8_48_1
## 141       126  R1       1     TMT36_R1 TMT36_R1      G5_0         1    G5_0_1
## 142       127  R2       1     TMT36_R2 TMT36_R2      G5_4         1    G5_4_1
## 143       128  R3       1     TMT36_R3 TMT36_R3     G5_24         1   G5_24_1
## 144       129  R4       1     TMT36_R4 TMT36_R4     G5_48         1   G5_48_1
## 145       126  R1       1     TMT37_R1 TMT37_R1      G1_0         1    G1_0_1
## 146       127  R2       1     TMT37_R2 TMT37_R2      G1_4         1    G1_4_1
## 147       128  R3       1     TMT37_R3 TMT37_R3     G1_24         1   G1_24_1
## 148       129  R4       1     TMT37_R4 TMT37_R4     G1_48         1   G1_48_1
## 149       126  R1       1     TMT38_R1 TMT38_R1      E5_0         1    E5_0_1
## 150       127  R2       1     TMT38_R2 TMT38_R2      E5_4         1    E5_4_1
## 151       128  R3       1     TMT38_R3 TMT38_R3     E5_24         1   E5_24_1
## 152       129  R4       1     TMT38_R4 TMT38_R4     E5_48         1   E5_48_1
## 153       126  R1       1     TMT39_R1 TMT39_R1      E4_0         1    E4_0_1
## 154       127  R2       1     TMT39_R2 TMT39_R2      E4_4         1    E4_4_1
## 155       128  R3       1     TMT39_R3 TMT39_R3     E4_24         1   E4_24_1
## 156       129  R4       1     TMT39_R4 TMT39_R4     E4_48         1   E4_48_1
##     EGFR_TKI_sen   measure
## 5      Sensitive Intensity
## 6      Sensitive Intensity
## 7      Sensitive Intensity
## 8      Sensitive Intensity
## 17     Sensitive Intensity
## 18     Sensitive Intensity
## 19     Sensitive Intensity
## 20     Sensitive Intensity
## 25     Sensitive Intensity
## 26     Sensitive Intensity
## 27     Sensitive Intensity
## 28     Sensitive Intensity
## 37     Sensitive Intensity
## 38     Sensitive Intensity
## 39     Sensitive Intensity
## 40     Sensitive Intensity
## 45     Sensitive Intensity
## 46     Sensitive Intensity
## 47     Sensitive Intensity
## 48     Sensitive Intensity
## 57     Sensitive Intensity
## 58     Sensitive Intensity
## 59     Sensitive Intensity
## 60     Sensitive Intensity
## 61     Resistant Intensity
## 62     Resistant Intensity
## 63     Resistant Intensity
## 64     Resistant Intensity
## 65     Resistant Intensity
## 66     Resistant Intensity
## 67     Resistant Intensity
## 68     Resistant Intensity
## 69     Resistant Intensity
## 70     Resistant Intensity
## 71     Resistant Intensity
## 72     Resistant Intensity
## 73     Resistant Intensity
## 74     Resistant Intensity
## 75     Resistant Intensity
## 76     Resistant Intensity
## 77     Resistant Intensity
## 78     Resistant Intensity
## 79     Resistant Intensity
## 80     Resistant Intensity
## 81     Resistant Intensity
## 82     Resistant Intensity
## 83     Resistant Intensity
## 84     Resistant Intensity
## 97     Resistant Intensity
## 98     Resistant Intensity
## 99     Resistant Intensity
## 100    Resistant Intensity
## 101    Resistant Intensity
## 102    Resistant Intensity
## 103    Resistant Intensity
## 104    Resistant Intensity
## 105    Resistant Intensity
## 106    Resistant Intensity
## 107    Resistant Intensity
## 108    Resistant Intensity
## 109    Resistant Intensity
## 110    Resistant Intensity
## 111    Resistant Intensity
## 112    Resistant Intensity
## 113    Resistant Intensity
## 114    Resistant Intensity
## 115    Resistant Intensity
## 116    Resistant Intensity
## 117    Resistant Intensity
## 118    Resistant Intensity
## 119    Resistant Intensity
## 120    Resistant Intensity
## 133    Resistant Intensity
## 134    Resistant Intensity
## 135    Resistant Intensity
## 136    Resistant Intensity
## 137    Resistant Intensity
## 138    Resistant Intensity
## 139    Resistant Intensity
## 140    Resistant Intensity
## 141    Resistant Intensity
## 142    Resistant Intensity
## 143    Resistant Intensity
## 144    Resistant Intensity
## 145    Resistant Intensity
## 146    Resistant Intensity
## 147    Resistant Intensity
## 148    Resistant Intensity
## 149    Resistant Intensity
## 150    Resistant Intensity
## 151    Resistant Intensity
## 152    Resistant Intensity
## 153    Resistant Intensity
## 154    Resistant Intensity
## 155    Resistant Intensity
## 156    Resistant Intensity

TMT_samples_Par <- subset(TMT_samples_all,TMT_samples_all$Cell.Line %in% c("C","CEv3"))

# Subset DEP object
DEP_TMT_Par <- se_mibs_TMT_bc[,paste0(TMT_samples_Par$name)]

# Save sample sheet
write.csv(TMT_samples_Par, file=paste0(output_dir,"DEP_TMT_Par",".csv"))

# Save DEP_2019_parental as RDS
saveRDS(DEP_TMT_Par, file = paste0(output_dir,format(Sys.Date(),"%y%m%d"),"_","DEP_TMT_Par",".rds"))

PCA

# PCA with all genes

# Create pca object
pcaData <- PCAtools::pca(assay(DEP_TMT_Par), metadata = colData(DEP_TMT_Par))

# Save pca data
write.csv(pcaData$rotated, file =paste0(output_dir,"pcaData",".csv"))

# Set colors
color_vector <-
    c(
    'C' = 'black',
    'CEv3' = 'red',
    'E4' = 'deepskyblue4',
    'E5' = 'turquoise3',
    'G1' = 'purple4',
    'G5' = 'mediumorchid',
    'G8' = 'deeppink',
    'G12' = 'pink2'
    )

# plot 5 pricipal components in a pairsplot
pairsplot <- pairsplot(pcaData,
    components = getComponents(pcaData, c(1:5)),
    triangle = TRUE, trianglelabSize = 12,
    hline = 0, vline = 0,
    pointSize = 0.4,
    gridlines.major = FALSE, gridlines.minor = FALSE,
    colby = 'Cell.Line',
    colkey= color_vector,
    title = 'Pairs plot', plotaxes = FALSE,
    legendPosition = 'none',
    margingaps = unit(c(-0.01, -0.01, -0.01, -0.01), 'cm'))

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pairsplot

# save graph as PDF
ggsave(
  filename = paste0(graph_dir,"pca_","pairsplot", ".pdf"),
  plot = pairsplot
  )

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# PCA 1
p1 <- biplot(pcaData, x = "PC1", y = "PC2",
       lab = NULL ,
       colby = "Cell.Line",
       colkey= color_vector,
       shape = "Timepoint",
       legendPosition = 'right' )

## Scale for colour is already present.
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p1

# save graph as PDF
ggsave(
  filename = paste0(graph_dir,"pca_vsd_500_","cell_line",".pdf"),
  plot = p1
  )

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Euclidian distances

# calculate sample euclidian distances
sampleDists <- dist(t(assay(DEP_TMT_Par)))

# plot sample euclidian distances via a heatmap
sampleDistMatrix <- as.matrix( sampleDists )

# Complex Heatmap annotation labels
annotation_col<-
  HeatmapAnnotation(df = data.frame(Model = DEP_TMT_Par@colData$Cell.Line,
                                    Time = DEP_TMT_Par@colData$Timepoint),
  col = list(Model =
  c(
    'C' = 'black',
    'CEv3' = 'red',
    'E4' = 'deepskyblue4',
    'E5' = 'turquoise3',
    'G1' = 'purple4',
    'G5' = 'mediumorchid',
    'G8' = 'deeppink',
    'G12' = 'pink2'
  ),
  Time =
  c(
    '0' = 'black',
    '4' = 'purple',
    '24' = 'darkgreen',
    '48' = 'darkorange'
  ))
  )

# Heatmap of euclidian distances
ComplexHeatmap::Heatmap(
      sampleDistMatrix,
      name = " ",
      column_title = "Eculidian Distances",
      show_column_names = FALSE,
      show_row_names = FALSE,
      clustering_distance_rows = "euclidean",
      clustering_distance_columns = "euclidean",
      clustering_method_rows = "complete",
      clustering_method_columns = "complete",
      col = colorRampPalette( rev(brewer.pal(9, "Blues")) )(255),
      top_annotation = annotation_col
    )

TVG (labeled)

# DEP_TMT_Par

# Directories 
dir.create(paste0(output_dir,"Heatmap_TVG"))
output_dir_Heatmap_TVG <- paste0(output_dir,"Heatmap_TVG/")
dir.create(paste0(graph_dir,"Heatmap_TVG"))
graph_dir_Heatmap_TVG <- paste0(graph_dir,"Heatmap_TVG/")

# pHeatmap of Top X DEG on transformed data
tvg <- c(100,50,25,10)
topVarGenesX_list <- list()

# pHeatmap of Top X DEG on transformed data

# Extract out distances
mat_list <- list()
for (i in seq_along(tvg)) {
  topVarGenesX_list[[paste0(tvg[i])]] <- head( order( rowVars( as.matrix(assay(DEP_TMT_Par)) ), decreasing=TRUE ), tvg[[i]] )
}
for (i in seq_along(tvg)) {
  mat_list[[paste0(tvg[i])]] <- assay(DEP_TMT_Par)[ topVarGenesX_list[[i]], ]
}

# Heatmap annotation labels
annotation_col<-
  HeatmapAnnotation(df = data.frame(Model = DEP_TMT_Par@colData$Cell.Line,
                                    Time = DEP_TMT_Par@colData$Timepoint),
  col = list(Model =
  c(
    'C' = 'black',
    'CEv3' = 'red',
    'E4' = 'deepskyblue4',
    'E5' = 'turquoise3',
    'G1' = 'purple4',
    'G5' = 'mediumorchid',
    'G8' = 'deeppink',
    'G12' = 'pink2'
  ),
  Time =
  c(
    '0' = 'black',
    '4' = 'purple',
    '24' = 'darkgreen',
    '48' = 'darkorange'
  ))
  )

# Generate Unlabeled Heatmap
p_list <- list()
for (i in seq_along(mat_list)) {
p_list[[paste0(names(mat_list)[[i]])]] <- ComplexHeatmap::Heatmap(
  t(scale(t(mat_list[[i]]))),
  top_annotation = annotation_col,
  name = " ",
  column_title = paste0(names(mat_list)[i],"_TVG"),
  show_column_names = F,
  show_row_names = F
  )
print(p_list[[i]])
}

# Save Heatmap
for (i in seq_along(p_list)) {
pdf(file=paste0(graph_dir_Heatmap_TVG, "CHeatmap_"  , names(p_list)[[i]], ".pdf"))
draw(p_list[[i]])
dev.off()
}


# Generate labeled Heatmap
p_list <- list()
for (i in seq_along(mat_list)) {
p_list[[paste0(names(mat_list)[[i]])]] <- ComplexHeatmap::Heatmap(
  t(scale(t(mat_list[[i]]))),
  #column_order=row.names(DEP_TMT_Par@colData[order(DEP_TMT_Par@colData$clid),]),
  top_annotation = annotation_col,
  column_split = factor(DEP_TMT_Par@colData$clid),
  name = " ",
  column_title = paste0(names(mat_list)[i],"_TVG"),
  show_column_names = F,
  show_row_names = T
  )
print(p_list[[i]])
}

# Save Heatmap
for (i in seq_along(p_list)) {
pdf(file=paste0(graph_dir_Heatmap_TVG, "CHeatmapLabel_"  , names(p_list)[[i]], ".pdf"))
draw(p_list[[i]])
dev.off()
}

# Graphics termination 
if(!is.null(dev.list())) dev.off()

## null device 
##           1

if(!is.null(dev.list())) graphics.off()

Limma

Comparisons

# Directories 
dir.create(paste0(output_dir,"Limma_res"))
output_dir_res <- paste0(output_dir,"Limma_res/")
dir.create(paste0(graph_dir,"Limma_res"))
graph_dir_res <- paste0(graph_dir,"Limma_res/")

# comps
comps_factor <- as.vector(factor(paste0(colData(DEP_TMT_Par)$condition)))
comps_df <- crossing(comp_num=comps_factor, comp_denom=comps_factor)
comps_df <- subset(comps_df, as.character(comps_df$comp_num) != as.character(comps_df$comp_denom))

# All comps
comps_df_all <- comps_df
comps_df_all <- cbind(comp_type="condition",comps_df_all)
comps_df_all <- cbind(comp_no=1:nrow(comps_df_all),comps_df_all)
comps_df_all$comp_name <- paste(comps_df_all$comp_num, "vs", comps_df_all$comp_denom, sep = "-")
comps_df_all$comp <- paste(comps_df_all$comp_num,comps_df_all$comp_denom, sep="-")
write.csv(comps_df_all, file=paste0(output_dir_res,"comps_all.csv"), col.names = T, row.names = F)

## Warning in write.csv(comps_df_all, file = paste0(output_dir_res,
## "comps_all.csv"), : attempt to set 'col.names' ignored

comps_all_subset <- comps_df_all[,2:4]

# Unique comps only (for only unique combo)
comps_df_unique <- comps_df[!duplicated(t(apply(comps_df, 1, sort))), ]
comps_df_unique <- cbind(comp_type="condition",comps_df_unique )
comps_df_unique <- cbind(comp_no=1:nrow(comps_df_unique),comps_df_unique)
comps_df_unique$comp_name <- paste(comps_df_unique$comp_num, "vs", comps_df_unique$comp_denom, sep = "-")
comps_df_unique$comp <- paste(comps_df_unique$comp_num,comps_df_unique$comp_denom, sep="-")
write.csv(comps_df_unique, file=paste0(output_dir_res,"comps_unique.csv"), col.names = T, row.names = F)

## Warning in write.csv(comps_df_unique, file = paste0(output_dir_res,
## "comps_unique.csv"), : attempt to set 'col.names' ignored

comps_unique_subset <- comps_df_unique[,2:4]

# Comp list to use (all comps)
comps <- comps_df_all

DE results

# Extract
# Results
# mibs_comp_list

# Directories 
dir.create(paste0(output_dir,"Limma_res"))

## Warning in dir.create(paste0(output_dir, "Limma_res")):
## '/Data/Output/MIB-DYN/DEP_TMT_Par/Limma_res' already exists

output_dir_res <- paste0(output_dir,"Limma_res/")
dir.create(paste0(graph_dir,"Limma_res"))

## Warning in dir.create(paste0(graph_dir, "Limma_res")):
## '/Data/Graphs/MIB-DYN/DEP_TMT_Par/Limma_res' already exists

graph_dir_res <- paste0(graph_dir,"Limma_res/")

# Extract results
DEP_TMT_limmaRes <- list()
for (i in seq_along(comps$comp)){
  DEP_TMT_limmaRes[[comps$comp[i]]] <-
    mibs_comp_list[[paste0(comps$comp[i])]]
}

# save results
for (i in seq_along(DEP_TMT_limmaRes)) {
  write.csv(
  DEP_TMT_limmaRes[[i]],
  file = paste0(output_dir_res,"comp_", i, "_", names(DEP_TMT_limmaRes)[i], ".csv"),
  row.names = TRUE
  )
}

Summarize Results

# Directories 
dir.create(paste0(output_dir,"Limma_res"))

## Warning in dir.create(paste0(output_dir, "Limma_res")):
## '/Data/Output/MIB-DYN/DEP_TMT_Par/Limma_res' already exists

output_dir_res <- paste0(output_dir,"Limma_res/")
dir.create(paste0(graph_dir,"Limma_res"))

## Warning in dir.create(paste0(graph_dir, "Limma_res")):
## '/Data/Graphs/MIB-DYN/DEP_TMT_Par/Limma_res' already exists

graph_dir_res <- paste0(graph_dir,"Limma_res/")

# Extract comps of interest
coi <- comps_df_all[comps_df_all$comp_denom=="C_0",]
comp_list_oi <- DEP_TMT_limmaRes[names(DEP_TMT_limmaRes) %in% coi$comp]
comp_list_oi <- lapply(comp_list_oi,data.frame)

# Label list of DF
for (i in seq_along(comp_list_oi)){
  colnames(comp_list_oi[[i]]) <- paste0(colnames(comp_list_oi[[i]]),"_",names(comp_list_oi)[[i]])
  comp_list_oi[[i]]$Gene_ID <- row.names(comp_list_oi[[i]])
}

# Left_join comps of interest to 1x spreadsheet
comp_list_oi_master <- Reduce(function(x, y) left_join(x, y, by="Gene_ID"), comp_list_oi)
row.names(comp_list_oi_master) <- comp_list_oi_master$Gene_ID
comp_list_oi_master <- comp_list_oi_master[,-which(colnames(comp_list_oi_master)=="Gene_ID")]
comp_list_oi_master <- comp_list_oi_master[,-which(colnames(comp_list_oi_master)%in% paste0(grep("gene_",colnames(comp_list_oi_master), value = T)))]

# Subset to spreadsheets
comp_list_oi_padj <- comp_list_oi_master[,grep("adj.P.Val_", colnames(comp_list_oi_master))]
comp_list_oi_pvalue <- comp_list_oi_master[,grep("P.Value_", colnames(comp_list_oi_master))]
comp_list_oi_l2fc <- comp_list_oi_master[,grep("logFC_", colnames(comp_list_oi_master))]

# Save spreadsheets
write.csv(comp_list_oi_padj, file = paste0(output_dir_res,"All_coi_","padj",".csv"))
write.csv(comp_list_oi_pvalue, file = paste0(output_dir_res,"All_coi_","pvalue",".csv"))
write.csv(comp_list_oi_l2fc, file = paste0(output_dir_res,"All_coi_","l2fc",".csv"))

Volcano Plots

# Directory for volcano plots
dir.create(paste0(graph_dir,"/","volcano"))
graph_dir_vol <- paste0(graph_dir,"volcano/")

# Plot Volcano
volcano_plots_ls <- list()
for (i in seq_along(DEP_TMT_limmaRes)){
  plot(volcano_plots_ls[[names(DEP_TMT_limmaRes)[i]]] <- 
    EnhancedVolcano(
      DEP_TMT_limmaRes[[i]], 
      lab=row.names(DEP_TMT_limmaRes[[i]]),
      x="logFC",
      y="adj.P.Val",
      title= paste0("MIBs ",names(DEP_TMT_limmaRes)[[i]]),
      pCutoff =qval, 
      FCcutoff =lfc,
      pointSize = 2.0, 
      labSize = 3.0 
    ))}

# Save Volcano
for (i in seq_along(volcano_plots_ls)){
ggsave(filename = paste0(graph_dir_vol,"comp_", i, "_", names(volcano_plots_ls)[[i]],".pdf"), plot=volcano_plots_ls[[i]])
}

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# Graphics termination 
if(!is.null(dev.list())) dev.off()

## null device 
##           1

if(!is.null(dev.list())) graphics.off()

DEP_TMT_BL_Res

Directories

# Directories for DEP_TMT_BL_Res
dir.create(paste0(MIB_graphs,"DEP_TMT_BL_Res"))
dir.create(paste0(MIB_output,"DEP_TMT_BL_Res"))
graph_dir <- paste0(MIB_graphs,"DEP_TMT_BL_Res/")
output_dir <- paste0(MIB_output,"DEP_TMT_BL_Res/")

Subset

# Subset 
TMT_samples_all

##        Prep   Run Number Sample Cell.Line clid Timepoint  serum Harvest
## 5   Prep_01 TMT02      6  P0298         C  701         0 starve  180807
## 6   Prep_01 TMT02      7  P0299         C  701         4 starve  180807
## 7   Prep_01 TMT02      8  P0300         C  701        24 starve  180808
## 8   Prep_01 TMT02      9  P0301         C  701        48 starve  180809
## 17  Prep_02 TMT05     21  P0294      CEv3  692         0 starve  180807
## 18  Prep_03 TMT05     22  P0295      CEv3  692         4 starve  180807
## 19  Prep_03 TMT05     23  P0296      CEv3  692        24 starve  180808
## 20  Prep_02 TMT05     24  P0297      CEv3  692        48 starve  180809
## 25  Prep_02 TMT07     31  P0117         C  701         0 starve  180705
## 26  Prep_02 TMT07     32  P0118         C  701         4 starve  180705
## 27  Prep_02 TMT07     33  P0119         C  701        24 starve  180706
## 28  Prep_02 TMT07     34  P0120         C  701        48 starve  180708
## 37  Prep_03 TMT10     46  P0290      CEv3  692         0 starve  180806
## 38  Prep_03 TMT10     47  P0291      CEv3  692         4 starve  180806
## 39  Prep_03 TMT10     48  P0292      CEv3  692        24 starve  180807
## 40  Prep_03 TMT10     49  P0293      CEv3  692        48 starve  180808
## 45  Prep_04 TMT12     56  P0113         C  701         0 starve  180704
## 46  Prep_04 TMT12     57  P0114         C  701         4 starve  180704
## 47  Prep_04 TMT12     58  P0115         C  701        24 starve  180705
## 48  Prep_04 TMT12     59  P0116         C  701        48 starve  180706
## 57  Prep_04 TMT15     71  P0286      CEv3  692         0 starve  180805
## 58  Prep_04 TMT15     72  P0287      CEv3  692         4 starve  180805
## 59  Prep_04 TMT15     73  P0288      CEv3  692        24 starve  180806
## 60  Prep_04 TMT15     74  P0289      CEv3  692        48 starve  180807
## 61  Prep_05 TMT16     76  P0368       G12  698         0 starve  180904
## 62  Prep_05 TMT16     77  P0369       G12  698         4 starve  180904
## 63  Prep_05 TMT16     78  P0370       G12  698        24 starve  180905
## 64  Prep_05 TMT16     79  P0371       G12  698        48 starve  180906
## 65  Prep_05 TMT17     81  P0356        G8  697         0 starve  180826
## 66  Prep_05 TMT17     82  P0357        G8  697         4 starve  180826
## 67  Prep_05 TMT17     83  P0358        G8  697        24 starve  180827
## 68  Prep_05 TMT17     84  P0359        G8  697        48 starve  180828
## 69  Prep_05 TMT18     86  P0344        G5  696         0 starve  180905
## 70  Prep_05 TMT18     87  P0345        G5  696         4 starve  180905
## 71  Prep_05 TMT18     88  P0346        G5  696        24 starve  180906
## 72  Prep_05 TMT18     89  P0347        G5  696        48 starve  180907
## 73  Prep_05 TMT19     91  P0332        G1  695         0 starve  180826
## 74  Prep_05 TMT19     92  P0333        G1  695         4 starve  180826
## 75  Prep_05 TMT19     93  P0334        G1  695        24 starve  180827
## 76  Prep_05 TMT19     94  P0335        G1  695        48 starve  180828
## 77  Prep_06 TMT20     96  P0204        E5  694         0 starve  180724
## 78  Prep_06 TMT20     97  P0205        E5  694         4 starve  180724
## 79  Prep_06 TMT20     98  P0206        E5  694        24 starve  180725
## 80  Prep_06 TMT20     99  P0207        E5  694        48 starve  180726
## 81  Prep_06 TMT21    101  P0189        E4  693         0 starve  180724
## 82  Prep_06 TMT21    102  P0190        E4  693         4 starve  180724
## 83  Prep_06 TMT21    103  P0191        E4  693        24 starve  180724
## 84  Prep_06 TMT21    104  P0192        E4  693        48 starve  180725
## 97  Prep_07 TMT25    121  P0364       G12  698         0 starve  180905
## 98  Prep_07 TMT25    122  P0365       G12  698         4 starve  180905
## 99  Prep_07 TMT25    123  P0366       G12  698        24 starve  180906
## 100 Prep_07 TMT25    124  P0367       G12  698        48 starve  180907
## 101 Prep_07 TMT26    126  P0352        G8  697         0 starve  180825
## 102 Prep_07 TMT26    127  P0353        G8  697         4 starve  180825
## 103 Prep_07 TMT26    128  P0354        G8  697        24 starve  180826
## 104 Prep_07 TMT26    129  P0355        G8  697        48 starve  180827
## 105 Prep_07 TMT27    131  P0340        G5  696         0 starve  180906
## 106 Prep_07 TMT27    132  P0341        G5  696         4 starve  180906
## 107 Prep_07 TMT27    133  P0342        G5  696        24 starve  180907
## 108 Prep_07 TMT27    134  P0343        G5  696        48 starve  180908
## 109 Prep_08 TMT28    136  P0328        G1  695         0 starve  180825
## 110 Prep_08 TMT28    137  P0329        G1  695         4 starve  180825
## 111 Prep_08 TMT28    138  P0330        G1  695        24 starve  180826
## 112 Prep_08 TMT28    139  P0331        G1  695        48 starve  180827
## 113 Prep_08 TMT29    141  P0200        E5  694         0 starve  180723
## 114 Prep_08 TMT29    142  P0201        E5  694         4 starve  180723
## 115 Prep_08 TMT29    143  P0202        E5  694        24 starve  180724
## 116 Prep_08 TMT29    144  P0203        E5  694        48 starve  180725
## 117 Prep_08 TMT30    146  P0185        E4  693         0 starve  180723
## 118 Prep_08 TMT30    147  P0186        E4  693         4 starve  180723
## 119 Prep_08 TMT30    148  P0187        E4  693        24 starve  180724
## 120 Prep_08 TMT30    149  P0188        E4  693        48 starve  180725
## 133 Prep_09 TMT34    166  P0360       G12  698         0 starve  180826
## 134 Prep_09 TMT34    167  P0361       G12  698         4 starve  180826
## 135 Prep_09 TMT34    168  P0362       G12  698        24 starve  180827
## 136 Prep_09 TMT34    169  P0363       G12  698        48 starve  180828
## 137 Prep_10 TMT35    171  P0348        G8  697         0 starve  180824
## 138 Prep_10 TMT35    172  P0349        G8  697         4 starve  180824
## 139 Prep_10 TMT35    173  P0350        G8  697        24 starve  180825
## 140 Prep_10 TMT35    174  P0351        G8  697        48 starve  180826
## 141 Prep_10 TMT36    176  P0336        G5  696         0 starve  180826
## 142 Prep_10 TMT36    177  P0337        G5  696         4 starve  180826
## 143 Prep_10 TMT36    178  P0338        G5  696        24 starve  180827
## 144 Prep_10 TMT36    179  P0339        G5  696        48 starve  180828
## 145 Prep_10 TMT37    181  P0324        G1  695         0 starve  180824
## 146 Prep_10 TMT37    182  P0325        G1  695         4 starve  180824
## 147 Prep_10 TMT37    183  P0326        G1  695        24 starve  180825
## 148 Prep_10 TMT37    184  P0327        G1  695        48 starve  180826
## 149 Prep_10 TMT38    186  P0196        E5  694         0 starve  180722
## 150 Prep_10 TMT38    187  P0197        E5  694         4 starve  180722
## 151 Prep_10 TMT38    188  P0198        E5  694        24 starve  180723
## 152 Prep_10 TMT38    189  P0199        E5  694        48 starve  180724
## 153 Prep_11 TMT39    191  P0181        E4  693         0 starve  180722
## 154 Prep_11 TMT39    192  P0182        E4  693         4 starve  180722
## 155 Prep_11 TMT39    193  P0183        E4  693        24 starve  180723
## 156 Prep_11 TMT39    194  P0184        E4  693        48 starve  180724
##     Label_num Tag Exp_rep TMT.Reporter    label condition replicate      name
## 5         126  R1       3     TMT02_R1 TMT02_R1       C_0         3     C_0_3
## 6         127  R2       3     TMT02_R2 TMT02_R2       C_4         3     C_4_3
## 7         128  R3       3     TMT02_R3 TMT02_R3      C_24         3    C_24_3
## 8         129  R4       3     TMT02_R4 TMT02_R4      C_48         3    C_48_3
## 17        126  R1       3     TMT05_R1 TMT05_R1    CEv3_0         3  CEv3_0_3
## 18        127  R2       3     TMT05_R2 TMT05_R2    CEv3_4         3  CEv3_4_3
## 19        128  R3       3     TMT05_R3 TMT05_R3   CEv3_24         3 CEv3_24_3
## 20        129  R4       3     TMT05_R4 TMT05_R4   CEv3_48         3 CEv3_48_3
## 25        126  R1       2     TMT07_R1 TMT07_R1       C_0         2     C_0_2
## 26        127  R2       2     TMT07_R2 TMT07_R2       C_4         2     C_4_2
## 27        128  R3       2     TMT07_R3 TMT07_R3      C_24         2    C_24_2
## 28        129  R4       2     TMT07_R4 TMT07_R4      C_48         2    C_48_2
## 37        126  R1       2     TMT10_R1 TMT10_R1    CEv3_0         2  CEv3_0_2
## 38        127  R2       2     TMT10_R2 TMT10_R2    CEv3_4         2  CEv3_4_2
## 39        128  R3       2     TMT10_R3 TMT10_R3   CEv3_24         2 CEv3_24_2
## 40        129  R4       2     TMT10_R4 TMT10_R4   CEv3_48         2 CEv3_48_2
## 45        126  R1       1     TMT12_R1 TMT12_R1       C_0         1     C_0_1
## 46        127  R2       1     TMT12_R2 TMT12_R2       C_4         1     C_4_1
## 47        128  R3       1     TMT12_R3 TMT12_R3      C_24         1    C_24_1
## 48        129  R4       1     TMT12_R4 TMT12_R4      C_48         1    C_48_1
## 57        126  R1       1     TMT15_R1 TMT15_R1    CEv3_0         1  CEv3_0_1
## 58        127  R2       1     TMT15_R2 TMT15_R2    CEv3_4         1  CEv3_4_1
## 59        128  R3       1     TMT15_R3 TMT15_R3   CEv3_24         1 CEv3_24_1
## 60        129  R4       1     TMT15_R4 TMT15_R4   CEv3_48         1 CEv3_48_1
## 61        126  R1       3     TMT16_R1 TMT16_R1     G12_0         3   G12_0_3
## 62        127  R2       3     TMT16_R2 TMT16_R2     G12_4         3   G12_4_3
## 63        128  R3       3     TMT16_R3 TMT16_R3    G12_24         3  G12_24_3
## 64        129  R4       3     TMT16_R4 TMT16_R4    G12_48         3  G12_48_3
## 65        126  R1       3     TMT17_R1 TMT17_R1      G8_0         3    G8_0_3
## 66        127  R2       3     TMT17_R2 TMT17_R2      G8_4         3    G8_4_3
## 67        128  R3       3     TMT17_R3 TMT17_R3     G8_24         3   G8_24_3
## 68        129  R4       3     TMT17_R4 TMT17_R4     G8_48         3   G8_48_3
## 69        126  R1       3     TMT18_R1 TMT18_R1      G5_0         3    G5_0_3
## 70        127  R2       3     TMT18_R2 TMT18_R2      G5_4         3    G5_4_3
## 71        128  R3       3     TMT18_R3 TMT18_R3     G5_24         3   G5_24_3
## 72        129  R4       3     TMT18_R4 TMT18_R4     G5_48         3   G5_48_3
## 73        126  R1       3     TMT19_R1 TMT19_R1      G1_0         3    G1_0_3
## 74        127  R2       3     TMT19_R2 TMT19_R2      G1_4         3    G1_4_3
## 75        128  R3       3     TMT19_R3 TMT19_R3     G1_24         3   G1_24_3
## 76        129  R4       3     TMT19_R4 TMT19_R4     G1_48         3   G1_48_3
## 77        126  R1       3     TMT20_R1 TMT20_R1      E5_0         3    E5_0_3
## 78        127  R2       3     TMT20_R2 TMT20_R2      E5_4         3    E5_4_3
## 79        128  R3       3     TMT20_R3 TMT20_R3     E5_24         3   E5_24_3
## 80        129  R4       3     TMT20_R4 TMT20_R4     E5_48         3   E5_48_3
## 81        126  R1       3     TMT21_R1 TMT21_R1      E4_0         3    E4_0_3
## 82        127  R2       3     TMT21_R2 TMT21_R2      E4_4         3    E4_4_3
## 83        128  R3       3     TMT21_R3 TMT21_R3     E4_24         3   E4_24_3
## 84        129  R4       3     TMT21_R4 TMT21_R4     E4_48         3   E4_48_3
## 97        126  R1       2     TMT25_R1 TMT25_R1     G12_0         2   G12_0_2
## 98        127  R2       2     TMT25_R2 TMT25_R2     G12_4         2   G12_4_2
## 99        128  R3       2     TMT25_R3 TMT25_R3    G12_24         2  G12_24_2
## 100       129  R4       2     TMT25_R4 TMT25_R4    G12_48         2  G12_48_2
## 101       126  R1       2     TMT26_R1 TMT26_R1      G8_0         2    G8_0_2
## 102       127  R2       2     TMT26_R2 TMT26_R2      G8_4         2    G8_4_2
## 103       128  R3       2     TMT26_R3 TMT26_R3     G8_24         2   G8_24_2
## 104       129  R4       2     TMT26_R4 TMT26_R4     G8_48         2   G8_48_2
## 105       126  R1       2     TMT27_R1 TMT27_R1      G5_0         2    G5_0_2
## 106       127  R2       2     TMT27_R2 TMT27_R2      G5_4         2    G5_4_2
## 107       128  R3       2     TMT27_R3 TMT27_R3     G5_24         2   G5_24_2
## 108       129  R4       2     TMT27_R4 TMT27_R4     G5_48         2   G5_48_2
## 109       126  R1       2     TMT28_R1 TMT28_R1      G1_0         2    G1_0_2
## 110       127  R2       2     TMT28_R2 TMT28_R2      G1_4         2    G1_4_2
## 111       128  R3       2     TMT28_R3 TMT28_R3     G1_24         2   G1_24_2
## 112       129  R4       2     TMT28_R4 TMT28_R4     G1_48         2   G1_48_2
## 113       126  R1       2     TMT29_R1 TMT29_R1      E5_0         2    E5_0_2
## 114       127  R2       2     TMT29_R2 TMT29_R2      E5_4         2    E5_4_2
## 115       128  R3       2     TMT29_R3 TMT29_R3     E5_24         2   E5_24_2
## 116       129  R4       2     TMT29_R4 TMT29_R4     E5_48         2   E5_48_2
## 117       126  R1       2     TMT30_R1 TMT30_R1      E4_0         2    E4_0_2
## 118       127  R2       2     TMT30_R2 TMT30_R2      E4_4         2    E4_4_2
## 119       128  R3       2     TMT30_R3 TMT30_R3     E4_24         2   E4_24_2
## 120       129  R4       2     TMT30_R4 TMT30_R4     E4_48         2   E4_48_2
## 133       126  R1       1     TMT34_R1 TMT34_R1     G12_0         1   G12_0_1
## 134       127  R2       1     TMT34_R2 TMT34_R2     G12_4         1   G12_4_1
## 135       128  R3       1     TMT34_R3 TMT34_R3    G12_24         1  G12_24_1
## 136       129  R4       1     TMT34_R4 TMT34_R4    G12_48         1  G12_48_1
## 137       126  R1       1     TMT35_R1 TMT35_R1      G8_0         1    G8_0_1
## 138       127  R2       1     TMT35_R2 TMT35_R2      G8_4         1    G8_4_1
## 139       128  R3       1     TMT35_R3 TMT35_R3     G8_24         1   G8_24_1
## 140       129  R4       1     TMT35_R4 TMT35_R4     G8_48         1   G8_48_1
## 141       126  R1       1     TMT36_R1 TMT36_R1      G5_0         1    G5_0_1
## 142       127  R2       1     TMT36_R2 TMT36_R2      G5_4         1    G5_4_1
## 143       128  R3       1     TMT36_R3 TMT36_R3     G5_24         1   G5_24_1
## 144       129  R4       1     TMT36_R4 TMT36_R4     G5_48         1   G5_48_1
## 145       126  R1       1     TMT37_R1 TMT37_R1      G1_0         1    G1_0_1
## 146       127  R2       1     TMT37_R2 TMT37_R2      G1_4         1    G1_4_1
## 147       128  R3       1     TMT37_R3 TMT37_R3     G1_24         1   G1_24_1
## 148       129  R4       1     TMT37_R4 TMT37_R4     G1_48         1   G1_48_1
## 149       126  R1       1     TMT38_R1 TMT38_R1      E5_0         1    E5_0_1
## 150       127  R2       1     TMT38_R2 TMT38_R2      E5_4         1    E5_4_1
## 151       128  R3       1     TMT38_R3 TMT38_R3     E5_24         1   E5_24_1
## 152       129  R4       1     TMT38_R4 TMT38_R4     E5_48         1   E5_48_1
## 153       126  R1       1     TMT39_R1 TMT39_R1      E4_0         1    E4_0_1
## 154       127  R2       1     TMT39_R2 TMT39_R2      E4_4         1    E4_4_1
## 155       128  R3       1     TMT39_R3 TMT39_R3     E4_24         1   E4_24_1
## 156       129  R4       1     TMT39_R4 TMT39_R4     E4_48         1   E4_48_1
##     EGFR_TKI_sen   measure
## 5      Sensitive Intensity
## 6      Sensitive Intensity
## 7      Sensitive Intensity
## 8      Sensitive Intensity
## 17     Sensitive Intensity
## 18     Sensitive Intensity
## 19     Sensitive Intensity
## 20     Sensitive Intensity
## 25     Sensitive Intensity
## 26     Sensitive Intensity
## 27     Sensitive Intensity
## 28     Sensitive Intensity
## 37     Sensitive Intensity
## 38     Sensitive Intensity
## 39     Sensitive Intensity
## 40     Sensitive Intensity
## 45     Sensitive Intensity
## 46     Sensitive Intensity
## 47     Sensitive Intensity
## 48     Sensitive Intensity
## 57     Sensitive Intensity
## 58     Sensitive Intensity
## 59     Sensitive Intensity
## 60     Sensitive Intensity
## 61     Resistant Intensity
## 62     Resistant Intensity
## 63     Resistant Intensity
## 64     Resistant Intensity
## 65     Resistant Intensity
## 66     Resistant Intensity
## 67     Resistant Intensity
## 68     Resistant Intensity
## 69     Resistant Intensity
## 70     Resistant Intensity
## 71     Resistant Intensity
## 72     Resistant Intensity
## 73     Resistant Intensity
## 74     Resistant Intensity
## 75     Resistant Intensity
## 76     Resistant Intensity
## 77     Resistant Intensity
## 78     Resistant Intensity
## 79     Resistant Intensity
## 80     Resistant Intensity
## 81     Resistant Intensity
## 82     Resistant Intensity
## 83     Resistant Intensity
## 84     Resistant Intensity
## 97     Resistant Intensity
## 98     Resistant Intensity
## 99     Resistant Intensity
## 100    Resistant Intensity
## 101    Resistant Intensity
## 102    Resistant Intensity
## 103    Resistant Intensity
## 104    Resistant Intensity
## 105    Resistant Intensity
## 106    Resistant Intensity
## 107    Resistant Intensity
## 108    Resistant Intensity
## 109    Resistant Intensity
## 110    Resistant Intensity
## 111    Resistant Intensity
## 112    Resistant Intensity
## 113    Resistant Intensity
## 114    Resistant Intensity
## 115    Resistant Intensity
## 116    Resistant Intensity
## 117    Resistant Intensity
## 118    Resistant Intensity
## 119    Resistant Intensity
## 120    Resistant Intensity
## 133    Resistant Intensity
## 134    Resistant Intensity
## 135    Resistant Intensity
## 136    Resistant Intensity
## 137    Resistant Intensity
## 138    Resistant Intensity
## 139    Resistant Intensity
## 140    Resistant Intensity
## 141    Resistant Intensity
## 142    Resistant Intensity
## 143    Resistant Intensity
## 144    Resistant Intensity
## 145    Resistant Intensity
## 146    Resistant Intensity
## 147    Resistant Intensity
## 148    Resistant Intensity
## 149    Resistant Intensity
## 150    Resistant Intensity
## 151    Resistant Intensity
## 152    Resistant Intensity
## 153    Resistant Intensity
## 154    Resistant Intensity
## 155    Resistant Intensity
## 156    Resistant Intensity

TMT_samples_BL_Res <- subset(TMT_samples_all,TMT_samples_all$Cell.Line %in% c("CEv3","E4","E5","G1","G5","G8","G12") & TMT_samples_all$Timepoint=="0" )

# Subset DEP object
DEP_TMT_BL_Res <- se_mibs_TMT_bc[,paste0(TMT_samples_BL_Res$name)]

# Save sample sheet
write.csv(TMT_samples_BL_Res, file=paste0(output_dir,"DEP_TMT_BL_Res",".csv"))

# Save DEP_2019_parental as RDS
saveRDS(DEP_TMT_BL_Res, file = paste0(output_dir,format(Sys.Date(),"%y%m%d"),"_","DEP_TMT_BL_Res",".rds"))

PCA

# PCA with all genes

# Create pca object
pcaData <- PCAtools::pca(assay(DEP_TMT_BL_Res), metadata = colData(DEP_TMT_BL_Res))

# Save pca data
write.csv(pcaData$rotated, file =paste0(output_dir,"pcaData",".csv"))

# Set colors
color_vector <-
    c(
    'C' = 'black',
    'CEv3' = 'red',
    'E4' = 'deepskyblue4',
    'E5' = 'turquoise3',
    'G1' = 'purple4',
    'G5' = 'mediumorchid',
    'G8' = 'deeppink',
    'G12' = 'pink2'
    )

# plot 5 pricipal components in a pairsplot
pairsplot <- pairsplot(pcaData,
    components = getComponents(pcaData, c(1:5)),
    triangle = TRUE, trianglelabSize = 12,
    hline = 0, vline = 0,
    pointSize = 0.4,
    gridlines.major = FALSE, gridlines.minor = FALSE,
    colby = 'Cell.Line',
    colkey= color_vector,
    title = 'Pairs plot', plotaxes = FALSE,
    legendPosition = 'none',
    margingaps = unit(c(-0.01, -0.01, -0.01, -0.01), 'cm'))

## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.

pairsplot

# save graph as PDF
ggsave(
  filename = paste0(graph_dir,"pca_","pairsplot", ".pdf"),
  plot = pairsplot
  )

## Saving 7 x 5 in image

# PCA 1
p1 <- biplot(pcaData, x = "PC1", y = "PC2",
       lab = NULL ,
       colby = "Cell.Line",
       colkey= color_vector,
       shape = "Timepoint",
       legendPosition = 'right' )

## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.

p1

# save graph as PDF
ggsave(
  filename = paste0(graph_dir,"pca_vsd_500_","cell_line",".pdf"),
  plot = p1
  )

## Saving 7 x 5 in image

Euclidian distances

# calculate sample euclidian distances
sampleDists <- dist(t(assay(DEP_TMT_BL_Res)))

# plot sample euclidian distances via a heatmap
sampleDistMatrix <- as.matrix( sampleDists )

# Complex Heatmap annotation labels
annotation_col<-
  HeatmapAnnotation(df = data.frame(Model = DEP_TMT_BL_Res@colData$Cell.Line,
                                    Time = DEP_TMT_BL_Res@colData$Timepoint),
  col = list(Model =
  c(
    'C' = 'black',
    'CEv3' = 'red',
    'E4' = 'deepskyblue4',
    'E5' = 'turquoise3',
    'G1' = 'purple4',
    'G5' = 'mediumorchid',
    'G8' = 'deeppink',
    'G12' = 'pink2'
  ),
  Time =
  c(
    '0' = 'black',
    '4' = 'purple',
    '24' = 'darkgreen',
    '48' = 'darkorange'
  ))
  )

# Heatmap of euclidian distances
ComplexHeatmap::Heatmap(
      sampleDistMatrix,
      name = " ",
      column_title = "Eculidian Distances",
      show_column_names = FALSE,
      show_row_names = FALSE,
      clustering_distance_rows = "euclidean",
      clustering_distance_columns = "euclidean",
      clustering_method_rows = "complete",
      clustering_method_columns = "complete",
      col = colorRampPalette( rev(brewer.pal(9, "Blues")) )(255),
      top_annotation = annotation_col
    )

TVG (labeled)

# DEP_TMT_BL_Res

# Directories 
dir.create(paste0(output_dir,"Heatmap_TVG"))
output_dir_Heatmap_TVG <- paste0(output_dir,"Heatmap_TVG/")
dir.create(paste0(graph_dir,"Heatmap_TVG"))
graph_dir_Heatmap_TVG <- paste0(graph_dir,"Heatmap_TVG/")

# pHeatmap of Top X DEG on transformed data
tvg <- c(100,50,25,10)
topVarGenesX_list <- list()

# pHeatmap of Top X DEG on transformed data

# Extract out distances
mat_list <- list()
for (i in seq_along(tvg)) {
  topVarGenesX_list[[paste0(tvg[i])]] <- head( order( rowVars( as.matrix(assay(DEP_TMT_BL_Res)) ), decreasing=TRUE ), tvg[[i]] )
}
for (i in seq_along(tvg)) {
  mat_list[[paste0(tvg[i])]] <- assay(DEP_TMT_BL_Res)[ topVarGenesX_list[[i]], ]
}

# Heatmap annotation labels
annotation_col<-
  HeatmapAnnotation(df = data.frame(Model = DEP_TMT_BL_Res@colData$Cell.Line,
                                    Time = DEP_TMT_BL_Res@colData$Timepoint),
  col = list(Model =
  c(
    'C' = 'black',
    'CEv3' = 'red',
    'E4' = 'deepskyblue4',
    'E5' = 'turquoise3',
    'G1' = 'purple4',
    'G5' = 'mediumorchid',
    'G8' = 'deeppink',
    'G12' = 'pink2'
  ),
  Time =
  c(
    '0' = 'black',
    '4' = 'purple',
    '24' = 'darkgreen',
    '48' = 'darkorange'
  ))
  )

# Generate Unlabeled Heatmap
p_list <- list()
for (i in seq_along(mat_list)) {
p_list[[paste0(names(mat_list)[[i]])]] <- ComplexHeatmap::Heatmap(
  t(scale(t(mat_list[[i]]))),
  top_annotation = annotation_col,
  name = " ",
  column_title = paste0(names(mat_list)[i],"_TVG"),
  show_column_names = F,
  show_row_names = F
  )
print(p_list[[i]])
}

# Save Heatmap
for (i in seq_along(p_list)) {
pdf(file=paste0(graph_dir_Heatmap_TVG, "CHeatmap_"  , names(p_list)[[i]], ".pdf"))
draw(p_list[[i]])
dev.off()
}


# Generate labeled Heatmap
p_list <- list()
for (i in seq_along(mat_list)) {
p_list[[paste0(names(mat_list)[[i]])]] <- ComplexHeatmap::Heatmap(
  t(scale(t(mat_list[[i]]))),
  #column_order=row.names(DEP_TMT_BL_Res@colData[order(DEP_TMT_BL_Res@colData$clid),]),
  top_annotation = annotation_col,
  column_split = factor(DEP_TMT_BL_Res@colData$clid),
  name = " ",
  column_title = paste0(names(mat_list)[i],"_TVG"),
  show_column_names = F,
  show_row_names = T
  )
print(p_list[[i]])
}

# Save Heatmap
for (i in seq_along(p_list)) {
pdf(file=paste0(graph_dir_Heatmap_TVG, "CHeatmapLabel_"  , names(p_list)[[i]], ".pdf"))
draw(p_list[[i]])
dev.off()
}

# Graphics termination 
if(!is.null(dev.list())) dev.off()

## null device 
##           1

if(!is.null(dev.list())) graphics.off()

Limma

Comparisons

# Directories 
dir.create(paste0(output_dir,"Limma_res"))
output_dir_res <- paste0(output_dir,"Limma_res/")
dir.create(paste0(graph_dir,"Limma_res"))
graph_dir_res <- paste0(graph_dir,"Limma_res/")

# comps
comps_factor <- as.vector(factor(paste0(colData(DEP_TMT_BL_Res)$condition)))
comps_df <- crossing(comp_num=comps_factor, comp_denom=comps_factor)
comps_df <- subset(comps_df, as.character(comps_df$comp_num) != as.character(comps_df$comp_denom))

# All comps
comps_df_all <- comps_df
comps_df_all <- cbind(comp_type="condition",comps_df_all)
comps_df_all <- cbind(comp_no=1:nrow(comps_df_all),comps_df_all)
comps_df_all$comp_name <- paste(comps_df_all$comp_num, "vs", comps_df_all$comp_denom, sep = "-")
comps_df_all$comp <- paste(comps_df_all$comp_num,comps_df_all$comp_denom, sep="-")
write.csv(comps_df_all, file=paste0(output_dir_res,"comps_all.csv"), col.names = T, row.names = F)

## Warning in write.csv(comps_df_all, file = paste0(output_dir_res,
## "comps_all.csv"), : attempt to set 'col.names' ignored

comps_all_subset <- comps_df_all[,2:4]

# Unique comps only (for only unique combo)
comps_df_unique <- comps_df[!duplicated(t(apply(comps_df, 1, sort))), ]
comps_df_unique <- cbind(comp_type="condition",comps_df_unique )
comps_df_unique <- cbind(comp_no=1:nrow(comps_df_unique),comps_df_unique)
comps_df_unique$comp_name <- paste(comps_df_unique$comp_num, "vs", comps_df_unique$comp_denom, sep = "-")
comps_df_unique$comp <- paste(comps_df_unique$comp_num,comps_df_unique$comp_denom, sep="-")
write.csv(comps_df_unique, file=paste0(output_dir_res,"comps_unique.csv"), col.names = T, row.names = F)

## Warning in write.csv(comps_df_unique, file = paste0(output_dir_res,
## "comps_unique.csv"), : attempt to set 'col.names' ignored

comps_unique_subset <- comps_df_unique[,2:4]

# Comp list to use (all comps)
comps <- comps_df_all

DE results

# Extract
# Results
# mibs_comp_list

# Directories 
dir.create(paste0(output_dir,"Limma_res"))

## Warning in dir.create(paste0(output_dir, "Limma_res")):
## '/Data/Output/MIB-DYN/DEP_TMT_BL_Res/Limma_res' already exists

output_dir_res <- paste0(output_dir,"Limma_res/")
dir.create(paste0(graph_dir,"Limma_res"))

## Warning in dir.create(paste0(graph_dir, "Limma_res")):
## '/Data/Graphs/MIB-DYN/DEP_TMT_BL_Res/Limma_res' already exists

graph_dir_res <- paste0(graph_dir,"Limma_res/")

# Extract results
DEP_TMT_limmaRes <- list()
for (i in seq_along(comps$comp)){
  DEP_TMT_limmaRes[[comps$comp[i]]] <-
    mibs_comp_list[[paste0(comps$comp[i])]]
}

# save results
for (i in seq_along(DEP_TMT_limmaRes)) {
  write.csv(
  DEP_TMT_limmaRes[[i]],
  file = paste0(output_dir_res,"comp_", i, "_", names(DEP_TMT_limmaRes)[i], ".csv"),
  row.names = TRUE
  )
}

Summarize Results

# Directories 
dir.create(paste0(output_dir,"Limma_res"))

## Warning in dir.create(paste0(output_dir, "Limma_res")):
## '/Data/Output/MIB-DYN/DEP_TMT_BL_Res/Limma_res' already exists

output_dir_res <- paste0(output_dir,"Limma_res/")
dir.create(paste0(graph_dir,"Limma_res"))

## Warning in dir.create(paste0(graph_dir, "Limma_res")):
## '/Data/Graphs/MIB-DYN/DEP_TMT_BL_Res/Limma_res' already exists

graph_dir_res <- paste0(graph_dir,"Limma_res/")

# Extract comps of interest
coi <- comps_df_all[comps_df_all$comp_denom=="CEv3_0",]
comp_list_oi <- DEP_TMT_limmaRes[names(DEP_TMT_limmaRes) %in% coi$comp]
comp_list_oi <- lapply(comp_list_oi,data.frame)

# Label list of DF
for (i in seq_along(comp_list_oi)){
  colnames(comp_list_oi[[i]]) <- paste0(colnames(comp_list_oi[[i]]),"_",names(comp_list_oi)[[i]])
  comp_list_oi[[i]]$Gene_ID <- row.names(comp_list_oi[[i]])
}

# Left_join comps of interest to 1x spreadsheet
comp_list_oi_master <- Reduce(function(x, y) left_join(x, y, by="Gene_ID"), comp_list_oi)
row.names(comp_list_oi_master) <- comp_list_oi_master$Gene_ID
comp_list_oi_master <- comp_list_oi_master[,-which(colnames(comp_list_oi_master)=="Gene_ID")]
comp_list_oi_master <- comp_list_oi_master[,-which(colnames(comp_list_oi_master)%in% paste0(grep("gene_",colnames(comp_list_oi_master), value = T)))]

# Subset to spreadsheets
comp_list_oi_padj <- comp_list_oi_master[,grep("adj.P.Val_", colnames(comp_list_oi_master))]
comp_list_oi_pvalue <- comp_list_oi_master[,grep("P.Value_", colnames(comp_list_oi_master))]
comp_list_oi_l2fc <- comp_list_oi_master[,grep("logFC_", colnames(comp_list_oi_master))]

# Save spreadsheets
write.csv(comp_list_oi_padj, file = paste0(output_dir_res,"All_coi_","padj",".csv"))
write.csv(comp_list_oi_pvalue, file = paste0(output_dir_res,"All_coi_","pvalue",".csv"))
write.csv(comp_list_oi_l2fc, file = paste0(output_dir_res,"All_coi_","l2fc",".csv"))

Volcano Plots

# Directory for volcano plots
dir.create(paste0(graph_dir,"/","volcano"))
graph_dir_vol <- paste0(graph_dir,"volcano/")

# Plot Volcano
volcano_plots_ls <- list()
for (i in seq_along(DEP_TMT_limmaRes)){
  plot(volcano_plots_ls[[names(DEP_TMT_limmaRes)[i]]] <- 
    EnhancedVolcano(
      DEP_TMT_limmaRes[[i]], 
      lab=row.names(DEP_TMT_limmaRes[[i]]),
      x="logFC",
      y="adj.P.Val",
      title= paste0("MIBs ",names(DEP_TMT_limmaRes)[[i]]),
      pCutoff =qval, 
      FCcutoff =lfc,
      pointSize = 2.0, 
      labSize = 3.0 
    ))}

# Save Volcano
for (i in seq_along(volcano_plots_ls)){
ggsave(filename = paste0(graph_dir_vol,"comp_", i, "_", names(volcano_plots_ls)[[i]],".pdf"), plot=volcano_plots_ls[[i]])
}

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# Graphics termination 
if(!is.null(dev.list())) dev.off()

## null device 
##           1

if(!is.null(dev.list())) graphics.off()

DEP_TMT_EDA

Directories

# Directories for DEP_TMT_EDA
dir.create(paste0(MIB_graphs,"DEP_TMT_EDA"))
dir.create(paste0(MIB_output,"DEP_TMT_EDA"))
graph_dir <- paste0(MIB_graphs,"DEP_TMT_EDA/")
output_dir <- paste0(MIB_output,"DEP_TMT_EDA/")

Subset

# Sample sheet
TMT_samples_all 

##        Prep   Run Number Sample Cell.Line clid Timepoint  serum Harvest
## 5   Prep_01 TMT02      6  P0298         C  701         0 starve  180807
## 6   Prep_01 TMT02      7  P0299         C  701         4 starve  180807
## 7   Prep_01 TMT02      8  P0300         C  701        24 starve  180808
## 8   Prep_01 TMT02      9  P0301         C  701        48 starve  180809
## 17  Prep_02 TMT05     21  P0294      CEv3  692         0 starve  180807
## 18  Prep_03 TMT05     22  P0295      CEv3  692         4 starve  180807
## 19  Prep_03 TMT05     23  P0296      CEv3  692        24 starve  180808
## 20  Prep_02 TMT05     24  P0297      CEv3  692        48 starve  180809
## 25  Prep_02 TMT07     31  P0117         C  701         0 starve  180705
## 26  Prep_02 TMT07     32  P0118         C  701         4 starve  180705
## 27  Prep_02 TMT07     33  P0119         C  701        24 starve  180706
## 28  Prep_02 TMT07     34  P0120         C  701        48 starve  180708
## 37  Prep_03 TMT10     46  P0290      CEv3  692         0 starve  180806
## 38  Prep_03 TMT10     47  P0291      CEv3  692         4 starve  180806
## 39  Prep_03 TMT10     48  P0292      CEv3  692        24 starve  180807
## 40  Prep_03 TMT10     49  P0293      CEv3  692        48 starve  180808
## 45  Prep_04 TMT12     56  P0113         C  701         0 starve  180704
## 46  Prep_04 TMT12     57  P0114         C  701         4 starve  180704
## 47  Prep_04 TMT12     58  P0115         C  701        24 starve  180705
## 48  Prep_04 TMT12     59  P0116         C  701        48 starve  180706
## 57  Prep_04 TMT15     71  P0286      CEv3  692         0 starve  180805
## 58  Prep_04 TMT15     72  P0287      CEv3  692         4 starve  180805
## 59  Prep_04 TMT15     73  P0288      CEv3  692        24 starve  180806
## 60  Prep_04 TMT15     74  P0289      CEv3  692        48 starve  180807
## 61  Prep_05 TMT16     76  P0368       G12  698         0 starve  180904
## 62  Prep_05 TMT16     77  P0369       G12  698         4 starve  180904
## 63  Prep_05 TMT16     78  P0370       G12  698        24 starve  180905
## 64  Prep_05 TMT16     79  P0371       G12  698        48 starve  180906
## 65  Prep_05 TMT17     81  P0356        G8  697         0 starve  180826
## 66  Prep_05 TMT17     82  P0357        G8  697         4 starve  180826
## 67  Prep_05 TMT17     83  P0358        G8  697        24 starve  180827
## 68  Prep_05 TMT17     84  P0359        G8  697        48 starve  180828
## 69  Prep_05 TMT18     86  P0344        G5  696         0 starve  180905
## 70  Prep_05 TMT18     87  P0345        G5  696         4 starve  180905
## 71  Prep_05 TMT18     88  P0346        G5  696        24 starve  180906
## 72  Prep_05 TMT18     89  P0347        G5  696        48 starve  180907
## 73  Prep_05 TMT19     91  P0332        G1  695         0 starve  180826
## 74  Prep_05 TMT19     92  P0333        G1  695         4 starve  180826
## 75  Prep_05 TMT19     93  P0334        G1  695        24 starve  180827
## 76  Prep_05 TMT19     94  P0335        G1  695        48 starve  180828
## 77  Prep_06 TMT20     96  P0204        E5  694         0 starve  180724
## 78  Prep_06 TMT20     97  P0205        E5  694         4 starve  180724
## 79  Prep_06 TMT20     98  P0206        E5  694        24 starve  180725
## 80  Prep_06 TMT20     99  P0207        E5  694        48 starve  180726
## 81  Prep_06 TMT21    101  P0189        E4  693         0 starve  180724
## 82  Prep_06 TMT21    102  P0190        E4  693         4 starve  180724
## 83  Prep_06 TMT21    103  P0191        E4  693        24 starve  180724
## 84  Prep_06 TMT21    104  P0192        E4  693        48 starve  180725
## 97  Prep_07 TMT25    121  P0364       G12  698         0 starve  180905
## 98  Prep_07 TMT25    122  P0365       G12  698         4 starve  180905
## 99  Prep_07 TMT25    123  P0366       G12  698        24 starve  180906
## 100 Prep_07 TMT25    124  P0367       G12  698        48 starve  180907
## 101 Prep_07 TMT26    126  P0352        G8  697         0 starve  180825
## 102 Prep_07 TMT26    127  P0353        G8  697         4 starve  180825
## 103 Prep_07 TMT26    128  P0354        G8  697        24 starve  180826
## 104 Prep_07 TMT26    129  P0355        G8  697        48 starve  180827
## 105 Prep_07 TMT27    131  P0340        G5  696         0 starve  180906
## 106 Prep_07 TMT27    132  P0341        G5  696         4 starve  180906
## 107 Prep_07 TMT27    133  P0342        G5  696        24 starve  180907
## 108 Prep_07 TMT27    134  P0343        G5  696        48 starve  180908
## 109 Prep_08 TMT28    136  P0328        G1  695         0 starve  180825
## 110 Prep_08 TMT28    137  P0329        G1  695         4 starve  180825
## 111 Prep_08 TMT28    138  P0330        G1  695        24 starve  180826
## 112 Prep_08 TMT28    139  P0331        G1  695        48 starve  180827
## 113 Prep_08 TMT29    141  P0200        E5  694         0 starve  180723
## 114 Prep_08 TMT29    142  P0201        E5  694         4 starve  180723
## 115 Prep_08 TMT29    143  P0202        E5  694        24 starve  180724
## 116 Prep_08 TMT29    144  P0203        E5  694        48 starve  180725
## 117 Prep_08 TMT30    146  P0185        E4  693         0 starve  180723
## 118 Prep_08 TMT30    147  P0186        E4  693         4 starve  180723
## 119 Prep_08 TMT30    148  P0187        E4  693        24 starve  180724
## 120 Prep_08 TMT30    149  P0188        E4  693        48 starve  180725
## 133 Prep_09 TMT34    166  P0360       G12  698         0 starve  180826
## 134 Prep_09 TMT34    167  P0361       G12  698         4 starve  180826
## 135 Prep_09 TMT34    168  P0362       G12  698        24 starve  180827
## 136 Prep_09 TMT34    169  P0363       G12  698        48 starve  180828
## 137 Prep_10 TMT35    171  P0348        G8  697         0 starve  180824
## 138 Prep_10 TMT35    172  P0349        G8  697         4 starve  180824
## 139 Prep_10 TMT35    173  P0350        G8  697        24 starve  180825
## 140 Prep_10 TMT35    174  P0351        G8  697        48 starve  180826
## 141 Prep_10 TMT36    176  P0336        G5  696         0 starve  180826
## 142 Prep_10 TMT36    177  P0337        G5  696         4 starve  180826
## 143 Prep_10 TMT36    178  P0338        G5  696        24 starve  180827
## 144 Prep_10 TMT36    179  P0339        G5  696        48 starve  180828
## 145 Prep_10 TMT37    181  P0324        G1  695         0 starve  180824
## 146 Prep_10 TMT37    182  P0325        G1  695         4 starve  180824
## 147 Prep_10 TMT37    183  P0326        G1  695        24 starve  180825
## 148 Prep_10 TMT37    184  P0327        G1  695        48 starve  180826
## 149 Prep_10 TMT38    186  P0196        E5  694         0 starve  180722
## 150 Prep_10 TMT38    187  P0197        E5  694         4 starve  180722
## 151 Prep_10 TMT38    188  P0198        E5  694        24 starve  180723
## 152 Prep_10 TMT38    189  P0199        E5  694        48 starve  180724
## 153 Prep_11 TMT39    191  P0181        E4  693         0 starve  180722
## 154 Prep_11 TMT39    192  P0182        E4  693         4 starve  180722
## 155 Prep_11 TMT39    193  P0183        E4  693        24 starve  180723
## 156 Prep_11 TMT39    194  P0184        E4  693        48 starve  180724
##     Label_num Tag Exp_rep TMT.Reporter    label condition replicate      name
## 5         126  R1       3     TMT02_R1 TMT02_R1       C_0         3     C_0_3
## 6         127  R2       3     TMT02_R2 TMT02_R2       C_4         3     C_4_3
## 7         128  R3       3     TMT02_R3 TMT02_R3      C_24         3    C_24_3
## 8         129  R4       3     TMT02_R4 TMT02_R4      C_48         3    C_48_3
## 17        126  R1       3     TMT05_R1 TMT05_R1    CEv3_0         3  CEv3_0_3
## 18        127  R2       3     TMT05_R2 TMT05_R2    CEv3_4         3  CEv3_4_3
## 19        128  R3       3     TMT05_R3 TMT05_R3   CEv3_24         3 CEv3_24_3
## 20        129  R4       3     TMT05_R4 TMT05_R4   CEv3_48         3 CEv3_48_3
## 25        126  R1       2     TMT07_R1 TMT07_R1       C_0         2     C_0_2
## 26        127  R2       2     TMT07_R2 TMT07_R2       C_4         2     C_4_2
## 27        128  R3       2     TMT07_R3 TMT07_R3      C_24         2    C_24_2
## 28        129  R4       2     TMT07_R4 TMT07_R4      C_48         2    C_48_2
## 37        126  R1       2     TMT10_R1 TMT10_R1    CEv3_0         2  CEv3_0_2
## 38        127  R2       2     TMT10_R2 TMT10_R2    CEv3_4         2  CEv3_4_2
## 39        128  R3       2     TMT10_R3 TMT10_R3   CEv3_24         2 CEv3_24_2
## 40        129  R4       2     TMT10_R4 TMT10_R4   CEv3_48         2 CEv3_48_2
## 45        126  R1       1     TMT12_R1 TMT12_R1       C_0         1     C_0_1
## 46        127  R2       1     TMT12_R2 TMT12_R2       C_4         1     C_4_1
## 47        128  R3       1     TMT12_R3 TMT12_R3      C_24         1    C_24_1
## 48        129  R4       1     TMT12_R4 TMT12_R4      C_48         1    C_48_1
## 57        126  R1       1     TMT15_R1 TMT15_R1    CEv3_0         1  CEv3_0_1
## 58        127  R2       1     TMT15_R2 TMT15_R2    CEv3_4         1  CEv3_4_1
## 59        128  R3       1     TMT15_R3 TMT15_R3   CEv3_24         1 CEv3_24_1
## 60        129  R4       1     TMT15_R4 TMT15_R4   CEv3_48         1 CEv3_48_1
## 61        126  R1       3     TMT16_R1 TMT16_R1     G12_0         3   G12_0_3
## 62        127  R2       3     TMT16_R2 TMT16_R2     G12_4         3   G12_4_3
## 63        128  R3       3     TMT16_R3 TMT16_R3    G12_24         3  G12_24_3
## 64        129  R4       3     TMT16_R4 TMT16_R4    G12_48         3  G12_48_3
## 65        126  R1       3     TMT17_R1 TMT17_R1      G8_0         3    G8_0_3
## 66        127  R2       3     TMT17_R2 TMT17_R2      G8_4         3    G8_4_3
## 67        128  R3       3     TMT17_R3 TMT17_R3     G8_24         3   G8_24_3
## 68        129  R4       3     TMT17_R4 TMT17_R4     G8_48         3   G8_48_3
## 69        126  R1       3     TMT18_R1 TMT18_R1      G5_0         3    G5_0_3
## 70        127  R2       3     TMT18_R2 TMT18_R2      G5_4         3    G5_4_3
## 71        128  R3       3     TMT18_R3 TMT18_R3     G5_24         3   G5_24_3
## 72        129  R4       3     TMT18_R4 TMT18_R4     G5_48         3   G5_48_3
## 73        126  R1       3     TMT19_R1 TMT19_R1      G1_0         3    G1_0_3
## 74        127  R2       3     TMT19_R2 TMT19_R2      G1_4         3    G1_4_3
## 75        128  R3       3     TMT19_R3 TMT19_R3     G1_24         3   G1_24_3
## 76        129  R4       3     TMT19_R4 TMT19_R4     G1_48         3   G1_48_3
## 77        126  R1       3     TMT20_R1 TMT20_R1      E5_0         3    E5_0_3
## 78        127  R2       3     TMT20_R2 TMT20_R2      E5_4         3    E5_4_3
## 79        128  R3       3     TMT20_R3 TMT20_R3     E5_24         3   E5_24_3
## 80        129  R4       3     TMT20_R4 TMT20_R4     E5_48         3   E5_48_3
## 81        126  R1       3     TMT21_R1 TMT21_R1      E4_0         3    E4_0_3
## 82        127  R2       3     TMT21_R2 TMT21_R2      E4_4         3    E4_4_3
## 83        128  R3       3     TMT21_R3 TMT21_R3     E4_24         3   E4_24_3
## 84        129  R4       3     TMT21_R4 TMT21_R4     E4_48         3   E4_48_3
## 97        126  R1       2     TMT25_R1 TMT25_R1     G12_0         2   G12_0_2
## 98        127  R2       2     TMT25_R2 TMT25_R2     G12_4         2   G12_4_2
## 99        128  R3       2     TMT25_R3 TMT25_R3    G12_24         2  G12_24_2
## 100       129  R4       2     TMT25_R4 TMT25_R4    G12_48         2  G12_48_2
## 101       126  R1       2     TMT26_R1 TMT26_R1      G8_0         2    G8_0_2
## 102       127  R2       2     TMT26_R2 TMT26_R2      G8_4         2    G8_4_2
## 103       128  R3       2     TMT26_R3 TMT26_R3     G8_24         2   G8_24_2
## 104       129  R4       2     TMT26_R4 TMT26_R4     G8_48         2   G8_48_2
## 105       126  R1       2     TMT27_R1 TMT27_R1      G5_0         2    G5_0_2
## 106       127  R2       2     TMT27_R2 TMT27_R2      G5_4         2    G5_4_2
## 107       128  R3       2     TMT27_R3 TMT27_R3     G5_24         2   G5_24_2
## 108       129  R4       2     TMT27_R4 TMT27_R4     G5_48         2   G5_48_2
## 109       126  R1       2     TMT28_R1 TMT28_R1      G1_0         2    G1_0_2
## 110       127  R2       2     TMT28_R2 TMT28_R2      G1_4         2    G1_4_2
## 111       128  R3       2     TMT28_R3 TMT28_R3     G1_24         2   G1_24_2
## 112       129  R4       2     TMT28_R4 TMT28_R4     G1_48         2   G1_48_2
## 113       126  R1       2     TMT29_R1 TMT29_R1      E5_0         2    E5_0_2
## 114       127  R2       2     TMT29_R2 TMT29_R2      E5_4         2    E5_4_2
## 115       128  R3       2     TMT29_R3 TMT29_R3     E5_24         2   E5_24_2
## 116       129  R4       2     TMT29_R4 TMT29_R4     E5_48         2   E5_48_2
## 117       126  R1       2     TMT30_R1 TMT30_R1      E4_0         2    E4_0_2
## 118       127  R2       2     TMT30_R2 TMT30_R2      E4_4         2    E4_4_2
## 119       128  R3       2     TMT30_R3 TMT30_R3     E4_24         2   E4_24_2
## 120       129  R4       2     TMT30_R4 TMT30_R4     E4_48         2   E4_48_2
## 133       126  R1       1     TMT34_R1 TMT34_R1     G12_0         1   G12_0_1
## 134       127  R2       1     TMT34_R2 TMT34_R2     G12_4         1   G12_4_1
## 135       128  R3       1     TMT34_R3 TMT34_R3    G12_24         1  G12_24_1
## 136       129  R4       1     TMT34_R4 TMT34_R4    G12_48         1  G12_48_1
## 137       126  R1       1     TMT35_R1 TMT35_R1      G8_0         1    G8_0_1
## 138       127  R2       1     TMT35_R2 TMT35_R2      G8_4         1    G8_4_1
## 139       128  R3       1     TMT35_R3 TMT35_R3     G8_24         1   G8_24_1
## 140       129  R4       1     TMT35_R4 TMT35_R4     G8_48         1   G8_48_1
## 141       126  R1       1     TMT36_R1 TMT36_R1      G5_0         1    G5_0_1
## 142       127  R2       1     TMT36_R2 TMT36_R2      G5_4         1    G5_4_1
## 143       128  R3       1     TMT36_R3 TMT36_R3     G5_24         1   G5_24_1
## 144       129  R4       1     TMT36_R4 TMT36_R4     G5_48         1   G5_48_1
## 145       126  R1       1     TMT37_R1 TMT37_R1      G1_0         1    G1_0_1
## 146       127  R2       1     TMT37_R2 TMT37_R2      G1_4         1    G1_4_1
## 147       128  R3       1     TMT37_R3 TMT37_R3     G1_24         1   G1_24_1
## 148       129  R4       1     TMT37_R4 TMT37_R4     G1_48         1   G1_48_1
## 149       126  R1       1     TMT38_R1 TMT38_R1      E5_0         1    E5_0_1
## 150       127  R2       1     TMT38_R2 TMT38_R2      E5_4         1    E5_4_1
## 151       128  R3       1     TMT38_R3 TMT38_R3     E5_24         1   E5_24_1
## 152       129  R4       1     TMT38_R4 TMT38_R4     E5_48         1   E5_48_1
## 153       126  R1       1     TMT39_R1 TMT39_R1      E4_0         1    E4_0_1
## 154       127  R2       1     TMT39_R2 TMT39_R2      E4_4         1    E4_4_1
## 155       128  R3       1     TMT39_R3 TMT39_R3     E4_24         1   E4_24_1
## 156       129  R4       1     TMT39_R4 TMT39_R4     E4_48         1   E4_48_1
##     EGFR_TKI_sen   measure
## 5      Sensitive Intensity
## 6      Sensitive Intensity
## 7      Sensitive Intensity
## 8      Sensitive Intensity
## 17     Sensitive Intensity
## 18     Sensitive Intensity
## 19     Sensitive Intensity
## 20     Sensitive Intensity
## 25     Sensitive Intensity
## 26     Sensitive Intensity
## 27     Sensitive Intensity
## 28     Sensitive Intensity
## 37     Sensitive Intensity
## 38     Sensitive Intensity
## 39     Sensitive Intensity
## 40     Sensitive Intensity
## 45     Sensitive Intensity
## 46     Sensitive Intensity
## 47     Sensitive Intensity
## 48     Sensitive Intensity
## 57     Sensitive Intensity
## 58     Sensitive Intensity
## 59     Sensitive Intensity
## 60     Sensitive Intensity
## 61     Resistant Intensity
## 62     Resistant Intensity
## 63     Resistant Intensity
## 64     Resistant Intensity
## 65     Resistant Intensity
## 66     Resistant Intensity
## 67     Resistant Intensity
## 68     Resistant Intensity
## 69     Resistant Intensity
## 70     Resistant Intensity
## 71     Resistant Intensity
## 72     Resistant Intensity
## 73     Resistant Intensity
## 74     Resistant Intensity
## 75     Resistant Intensity
## 76     Resistant Intensity
## 77     Resistant Intensity
## 78     Resistant Intensity
## 79     Resistant Intensity
## 80     Resistant Intensity
## 81     Resistant Intensity
## 82     Resistant Intensity
## 83     Resistant Intensity
## 84     Resistant Intensity
## 97     Resistant Intensity
## 98     Resistant Intensity
## 99     Resistant Intensity
## 100    Resistant Intensity
## 101    Resistant Intensity
## 102    Resistant Intensity
## 103    Resistant Intensity
## 104    Resistant Intensity
## 105    Resistant Intensity
## 106    Resistant Intensity
## 107    Resistant Intensity
## 108    Resistant Intensity
## 109    Resistant Intensity
## 110    Resistant Intensity
## 111    Resistant Intensity
## 112    Resistant Intensity
## 113    Resistant Intensity
## 114    Resistant Intensity
## 115    Resistant Intensity
## 116    Resistant Intensity
## 117    Resistant Intensity
## 118    Resistant Intensity
## 119    Resistant Intensity
## 120    Resistant Intensity
## 133    Resistant Intensity
## 134    Resistant Intensity
## 135    Resistant Intensity
## 136    Resistant Intensity
## 137    Resistant Intensity
## 138    Resistant Intensity
## 139    Resistant Intensity
## 140    Resistant Intensity
## 141    Resistant Intensity
## 142    Resistant Intensity
## 143    Resistant Intensity
## 144    Resistant Intensity
## 145    Resistant Intensity
## 146    Resistant Intensity
## 147    Resistant Intensity
## 148    Resistant Intensity
## 149    Resistant Intensity
## 150    Resistant Intensity
## 151    Resistant Intensity
## 152    Resistant Intensity
## 153    Resistant Intensity
## 154    Resistant Intensity
## 155    Resistant Intensity
## 156    Resistant Intensity

# Subset by cell line
TMT_samples_EDA_ls <- list()
for ( i in unique(TMT_samples_all$Cell.Line)){
  TMT_samples_EDA_ls[[i]] <-
  subset(TMT_samples_all, TMT_samples_all$Cell.Line==paste0(i))
}

# Add additional groups 
TMT_samples_EDA_ls <- c(TMT_samples_EDA_ls,
                        list(
                          TMT_samples_BL_Res=TMT_samples_BL_Res,
                          TMT_samples_Res=TMT_samples_Res,
                          TMT_samples_Par=TMT_samples_Par
                        ))


# Save sample sheet
for (i in seq_along(TMT_samples_EDA_ls)){
  dir.create(paste0(graph_dir,names(TMT_samples_EDA_ls)[i]))
  dir.create(paste0(output_dir,names(TMT_samples_EDA_ls)[i]))
  write.csv(TMT_samples_EDA_ls[[i]], file=paste0(output_dir,names(TMT_samples_EDA_ls)[i],"/TMT_samples",".csv"))
}

# Imputed values for TMT
se_mibs_TMT_mv

## class: SummarizedExperiment 
## dim: 306 96 
## metadata(0):
## assays(1): ''
## rownames(306): Ephb6 Clk2 ... Riok3 Pkmyt1
## rowData names(4): ID name imputed num_NAs
## colnames(96): C_0_3 C_4_3 ... E4_24_1 E4_48_1
## colData names(20): Prep Run ... EGFR_TKI_sen measure

# Save as RDS
saveRDS(se_mibs_TMT_mv, file = paste0(output_dir,format(Sys.Date(),"%y%m%d"),"_","se_mibs_TMT_mv",".rds"))

# Batch corrected values for TMT
se_mibs_TMT_bc

## class: SummarizedExperiment 
## dim: 306 96 
## metadata(0):
## assays(1): ''
## rownames(306): Ephb6 Clk2 ... Riok3 Pkmyt1
## rowData names(4): ID name imputed num_NAs
## colnames(96): C_0_3 C_4_3 ... E4_24_1 E4_48_1
## colData names(20): Prep Run ... EGFR_TKI_sen measure

# Save as RDS
saveRDS(se_mibs_TMT_bc, file = paste0(output_dir,format(Sys.Date(),"%y%m%d"),"_","se_mibs_TMT_bc",".rds"))

# Generate se subsets for DEP EDA
DEP_TMT_EDA_mv_ls <- list()
DEP_TMT_EDA_bc_ls <- list()
for (i in seq_along(TMT_samples_EDA_ls)){
  
  # Generate se object for imputed missing values
  DEP_TMT_EDA_mv_ls[[names(TMT_samples_EDA_ls)[i]]] <-
    se_mibs_TMT_mv[,paste0(TMT_samples_EDA_ls[[i]]$condition,"_",TMT_samples_EDA_ls[[i]]$replicate)]
  # Save as RDS
  saveRDS(DEP_TMT_EDA_mv_ls[[names(TMT_samples_EDA_ls)[i]]], file = paste0(output_dir,names(TMT_samples_EDA_ls)[i],"/",format(Sys.Date(),"%y%m%d"),"_",names(TMT_samples_EDA_ls)[i],"_mv_",".rds"))
 
   # Generate se object for batch corrected values
  DEP_TMT_EDA_bc_ls[[names(TMT_samples_EDA_ls)[i]]] <-
    se_mibs_TMT_bc[,paste0(TMT_samples_EDA_ls[[i]]$condition,"_",TMT_samples_EDA_ls[[i]]$replicate)]
  # Save as RDS
  saveRDS(DEP_TMT_EDA_bc_ls[[names(TMT_samples_EDA_ls)[i]]], file = paste0(output_dir,names(TMT_samples_EDA_ls)[i],"/",format(Sys.Date(),"%y%m%d"),"_",names(TMT_samples_EDA_ls)[i],"_bc_",".rds"))
  
  # Save imputed(raw) and batch corrected (norm) norm counts
  norm_counts <- as.data.frame(assay(DEP_TMT_EDA_bc_ls[[names(TMT_samples_EDA_ls)[i]]]))
  raw_counts <- as.data.frame(assay(DEP_TMT_EDA_mv_ls[[names(TMT_samples_EDA_ls)[i]]]))

  write.csv(
  norm_counts,
  file=paste0(output_dir,names(TMT_samples_EDA_ls)[i],"/","norm_counts",".csv"),
  row.names = TRUE
  )

  write.csv(
  raw_counts,
  file=paste0(output_dir,names(TMT_samples_EDA_ls)[i],"/","raw_counts",".csv"),
  row.names = TRUE
  )
}

# Save as RDS
saveRDS(DEP_TMT_EDA_mv_ls, file = paste0(output_dir,format(Sys.Date(),"%y%m%d"),"_","DEP_TMT_EDA_mv_ls",".rds"))

# Save as RDS
saveRDS(DEP_TMT_EDA_bc_ls, file = paste0(output_dir,format(Sys.Date(),"%y%m%d"),"_","DEP_TMT_EDA_bc_ls",".rds"))

PCA

# PCA with all genes

# BC PCA
for(h in seq_along(DEP_TMT_EDA_bc_ls)){
  
  # Directories
  PCA_graph <- paste0(graph_dir,names(DEP_TMT_EDA_bc_ls)[h],"/")
  PCA_output <- paste0(output_dir,names(DEP_TMT_EDA_bc_ls)[h],"/")
  
  # Extract single
  DEP_bc <- DEP_TMT_EDA_bc_ls[[h]]
  
  # Create pca object
  pcaData <- PCAtools::pca(assay(DEP_bc), metadata = colData(DEP_bc))
  
  # Save pca data
  write.csv(pcaData$rotated, file =paste0(PCA_output,"pcaData","DEP_bc",".csv"))
  
  # Set colors
color_vector <-
  c(
    '0' = 'black',
    '4' = 'purple',
    '24' = 'darkgreen',
    '48' = 'darkorange'
  )

  # plot 5 pricipal components in a pairsplot
  pairsplot <- pairsplot(pcaData,
      components = getComponents(pcaData, c(1:5)),
      triangle = TRUE, trianglelabSize = 12,
      hline = 0, vline = 0,
      pointSize = 0.4,
      gridlines.major = FALSE, gridlines.minor = FALSE,
      colby = 'Timepoint',
      colkey= color_vector,
      title = 'Pairs plot', plotaxes = FALSE,
      legendPosition = 'none',
      margingaps = unit(c(-0.01, -0.01, -0.01, -0.01), 'cm'))
  pairsplot
  # save graph as PDF
  ggsave(
    filename = paste0(PCA_graph,"DEP_bc_","pairsplot", ".pdf"),
    plot = pairsplot
    )
  
  # PCA 1
  p1 <- biplot(pcaData, x = "PC1", y = "PC2",
         lab = NULL ,
         colby = "Timepoint",
         colkey= color_vector,
         legendPosition = 'right' )
  p1
  # save graph as PDF
  ggsave(
    filename = paste0(PCA_graph,"pca_DEP_bc_","Timepoint",".pdf"),
    plot = p1
    )
  
}

## Scale for colour is already present.
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## Coordinate system already present. Adding new coordinate system, which will
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## Saving 7 x 5 in image
## Scale for colour is already present.
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## Saving 7 x 5 in image
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## Scale for colour is already present.
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## Scale for colour is already present.
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## Scale for colour is already present.
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## Scale for colour is already present.
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## Scale for colour is already present.
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## Scale for colour is already present.
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## Scale for colour is already present.
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## Scale for colour is already present.
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## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
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## Coordinate system already present. Adding new coordinate system, which will
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## Coordinate system already present. Adding new coordinate system, which will
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## Coordinate system already present. Adding new coordinate system, which will
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## Coordinate system already present. Adding new coordinate system, which will
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## Coordinate system already present. Adding new coordinate system, which will
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## Coordinate system already present. Adding new coordinate system, which will
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## Coordinate system already present. Adding new coordinate system, which will
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## Coordinate system already present. Adding new coordinate system, which will
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## Coordinate system already present. Adding new coordinate system, which will
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## Coordinate system already present. Adding new coordinate system, which will
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## Coordinate system already present. Adding new coordinate system, which will
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## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Saving 7 x 5 in image
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Saving 7 x 5 in image
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
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## Coordinate system already present. Adding new coordinate system, which will
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## Coordinate system already present. Adding new coordinate system, which will
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## Coordinate system already present. Adding new coordinate system, which will
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## Coordinate system already present. Adding new coordinate system, which will
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## Coordinate system already present. Adding new coordinate system, which will
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## Coordinate system already present. Adding new coordinate system, which will
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## Coordinate system already present. Adding new coordinate system, which will
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## Coordinate system already present. Adding new coordinate system, which will
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## Coordinate system already present. Adding new coordinate system, which will
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## Coordinate system already present. Adding new coordinate system, which will
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## Coordinate system already present. Adding new coordinate system, which will
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## Coordinate system already present. Adding new coordinate system, which will
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## Coordinate system already present. Adding new coordinate system, which will
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## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Saving 7 x 5 in image
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Saving 7 x 5 in image
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
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## Coordinate system already present. Adding new coordinate system, which will
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## Coordinate system already present. Adding new coordinate system, which will
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## Coordinate system already present. Adding new coordinate system, which will
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## Coordinate system already present. Adding new coordinate system, which will
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## Coordinate system already present. Adding new coordinate system, which will
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## Coordinate system already present. Adding new coordinate system, which will
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## Coordinate system already present. Adding new coordinate system, which will
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## Coordinate system already present. Adding new coordinate system, which will
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## Coordinate system already present. Adding new coordinate system, which will
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## Coordinate system already present. Adding new coordinate system, which will
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## Coordinate system already present. Adding new coordinate system, which will
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## Coordinate system already present. Adding new coordinate system, which will
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## Coordinate system already present. Adding new coordinate system, which will
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## Coordinate system already present. Adding new coordinate system, which will
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## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Saving 7 x 5 in image
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Saving 7 x 5 in image
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Saving 7 x 5 in image
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Saving 7 x 5 in image
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Saving 7 x 5 in image
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Saving 7 x 5 in image
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Saving 7 x 5 in image
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Saving 7 x 5 in image
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Saving 7 x 5 in image
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Saving 7 x 5 in image
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Saving 7 x 5 in image
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Saving 7 x 5 in image
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Saving 7 x 5 in image
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Saving 7 x 5 in image
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Saving 7 x 5 in image
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Saving 7 x 5 in image

# Missing Values PCA
for(h in seq_along(DEP_TMT_EDA_mv_ls)){
  
  # Directories
  PCA_graph <- paste0(graph_dir,names(DEP_TMT_EDA_mv_ls)[h],"/")
  PCA_output <- paste0(output_dir,names(DEP_TMT_EDA_mv_ls)[h],"/")
  
  # Extract single
  DEP_mv <- DEP_TMT_EDA_mv_ls[[h]]
  
  # Create pca object
  pcaData <- PCAtools::pca(assay(DEP_mv), metadata = colData(DEP_mv))
  
  # Save pca data
  write.csv(pcaData$rotated, file =paste0(PCA_output,"pcaData","DEP_mv",".csv"))
  
  # Set colors
color_vector <-
  c(
    '0' = 'black',
    '4' = 'purple',
    '24' = 'darkgreen',
    '48' = 'darkorange'
  )
  
  # plot 5 pricipal components in a pairsplot
  pairsplot <- pairsplot(pcaData,
      components = getComponents(pcaData, c(1:5)),
      triangle = TRUE, trianglelabSize = 12,
      hline = 0, vline = 0,
      pointSize = 0.4,
      gridlines.major = FALSE, gridlines.minor = FALSE,
      colby = 'Timepoint',
      colkey= color_vector,
      title = 'Pairs plot', plotaxes = FALSE,
      legendPosition = 'none',
      margingaps = unit(c(-0.01, -0.01, -0.01, -0.01), 'cm'))
  pairsplot
  # save graph as PDF
  ggsave(
    filename = paste0(PCA_graph,"DEP_mv","pairsplot", ".pdf"),
    plot = pairsplot
    )
  
  # PCA 1
  p1 <- biplot(pcaData, x = "PC1", y = "PC2",
         lab = NULL ,
         colby = "Timepoint",
         colkey= color_vector,
         legendPosition = 'right' )
  p1
  # save graph as PDF
  ggsave(
    filename = paste0(PCA_graph,"pca_DEP_mv_","Timepoint",".pdf"),
    plot = p1
    )
  
}

## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Saving 7 x 5 in image
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Saving 7 x 5 in image
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Saving 7 x 5 in image
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Saving 7 x 5 in image
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Saving 7 x 5 in image
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Saving 7 x 5 in image
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Saving 7 x 5 in image
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Saving 7 x 5 in image
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Saving 7 x 5 in image
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Saving 7 x 5 in image
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Saving 7 x 5 in image
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Saving 7 x 5 in image
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Saving 7 x 5 in image
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Saving 7 x 5 in image
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Saving 7 x 5 in image
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Saving 7 x 5 in image
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Saving 7 x 5 in image
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Saving 7 x 5 in image
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Coordinate system already present. Adding new coordinate system, which will
## replace the existing one.
## Saving 7 x 5 in image
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Saving 7 x 5 in image
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
## Scale for colour is already present.
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Euclidian distances

Batch corrected values

system.time(
for(h in seq_along(DEP_TMT_EDA_bc_ls)){
  
  # Directories
  Euclidian_graph <- paste0(graph_dir,names(DEP_TMT_EDA_bc_ls)[h],"/")
  Euclidian_output <- paste0(output_dir,names(DEP_TMT_EDA_bc_ls)[h],"/")
  
  # Extract single
  DEP_bc <- DEP_TMT_EDA_bc_ls[[h]]

# calculate sample euclidian distances
sampleDists <- dist(t(assay(DEP_bc)))

# plot sample euclidian distances via a heatmap
sampleDistMatrix <- as.matrix( sampleDists )

# Complex Heatmap annotation labels
annotation_col<-
  HeatmapAnnotation(df = data.frame(Model = DEP_bc@colData$Cell.Line,
                                    Time = DEP_bc@colData$Timepoint),
  col = list(Model =
  c(
    'C' = 'black',
    'CEv3' = 'red',
    'E4' = 'deepskyblue4',
    'E5' = 'turquoise3',
    'G1' = 'purple4',
    'G5' = 'mediumorchid',
    'G8' = 'deeppink',
    'G12' = 'pink2'
  ),
  Time =
    c(
    '0' = 'black',
    '4' = 'purple',
    '24' = 'darkgreen',
    '48' = 'darkorange'
      )
  ))

# Heatmap of euclidian distances
ed_heatmap <-
  ComplexHeatmap::Heatmap(
      sampleDistMatrix,
      name = " ",
      column_title = "Euclidian Distances",
      show_column_names = FALSE,
      show_row_names = FALSE,
      clustering_distance_rows = "euclidean",
      clustering_distance_columns = "euclidean",
      clustering_method_rows = "complete",
      clustering_method_columns = "complete",
      col = colorRampPalette( rev(brewer.pal(9, "Blues")) )(255),
      top_annotation = annotation_col
    )

pdf(file=paste0(Euclidian_graph,"Euclidian_distance_bc",".pdf"))
draw(ed_heatmap)
dev.off()

}
)

##    user  system elapsed 
##   2.574   0.000   2.668

Missing Values

system.time(
for(h in seq_along(DEP_TMT_EDA_mv_ls)){
  
  # Directories
  Euclidian_graph <- paste0(graph_dir,names(DEP_TMT_EDA_mv_ls)[h],"/")
  Euclidian_output <- paste0(output_dir,names(DEP_TMT_EDA_mv_ls)[h],"/")
  
  # Extract single
  DEP_mv <- DEP_TMT_EDA_mv_ls[[h]]

# calculate sample euclidian distances
sampleDists <- dist(t(assay(DEP_mv)))

# plot sample euclidian distances via a heatmap
sampleDistMatrix <- as.matrix( sampleDists )

# Complex Heatmap annotation labels
annotation_col<-
  HeatmapAnnotation(df = data.frame(Model = DEP_mv@colData$Cell.Line,
                                    Time = DEP_mv@colData$Timepoint),
  col = list(Model =
  c(
    'C' = 'black',
    'CEv3' = 'red',
    'E4' = 'deepskyblue4',
    'E5' = 'turquoise3',
    'G1' = 'purple4',
    'G5' = 'mediumorchid',
    'G8' = 'deeppink',
    'G12' = 'pink2'
  ),
  Time =
    c(
    '0' = 'black',
    '4' = 'purple',
    '24' = 'darkgreen',
    '48' = 'darkorange'
      )
  ))

# Heatmap of euclidian distances
ed_heatmap <-
  ComplexHeatmap::Heatmap(
      sampleDistMatrix,
      name = " ",
      column_title = "Euclidian Distances",
      show_column_names = FALSE,
      show_row_names = FALSE,
      clustering_distance_rows = "euclidean",
      clustering_distance_columns = "euclidean",
      clustering_method_rows = "complete",
      clustering_method_columns = "complete",
      col = colorRampPalette( rev(brewer.pal(9, "Blues")) )(255),
      top_annotation = annotation_col
    )

pdf(file=paste0(Euclidian_graph,"Euclidian_distance_mv",".pdf"))
draw(ed_heatmap)
dev.off()

}
)

##    user  system elapsed 
##   2.615   0.010   2.911

Genes of Interest (GOI)

This chunk will extract the normalized batch corrected counts for GOI ##### normCounts

#  user  system elapsed 
# 602.754   5.045 732.333 

system.time(for (h in seq_along(DEP_TMT_EDA_bc_ls)){

# Directories for normCounts
dir.create(paste0(output_dir,names(DEP_TMT_EDA_bc_ls)[h],"/","normCounts"))
output_dir_normCounts <- paste0(output_dir,names(DEP_TMT_EDA_bc_ls)[h],"/","normCounts/")
dir.create(paste0(graph_dir,names(DEP_TMT_EDA_bc_ls)[h],"/","normCounts"))
graph_dir_normCounts <- paste0(graph_dir,names(DEP_TMT_EDA_bc_ls)[h],"/","normCounts/")

DEP_normCounts <- DEP_TMT_EDA_bc_ls[[h]]

# DEP_normCounts
norm_counts <- as.data.frame(assay(DEP_normCounts))
normCounts <- norm_counts

# Import DF of GOI
# normCounts_goi_df <- read.csv("/Data/Input/RNAseq/REF/normCounts_goi_mouse.csv",header = TRUE, fileEncoding = "UTF-8-BOM")

# vectors of genes of interest. Filtered for present
goi <- row.names(DEP_normCounts)

# Check all goi are present after filtering
match(goi,row.names(normCounts))

# Transform data
counts_goi <- as.data.frame(t(normCounts[goi,]))
counts.rn_counts_goi <- rownames(counts_goi)

# Extract each gene in to a unique dataframe
counts_data_list_goi <- list()
for (i in seq_along(goi)) {
  counts_data_list_goi[[goi[i]]] <- as.data.frame(counts_goi[, i])
  rownames(counts_data_list_goi[[goi[i]]]) <- counts.rn_counts_goi
  counts_data_list_goi[[goi[i]]] <-
    rownames_to_column(counts_data_list_goi[[goi[i]]], var = "sample_id")
  colnames(counts_data_list_goi[[goi[i]]])[2] <- "norm_counts"
  counts_data_list_goi[[goi[i]]]$cell_line_abbr <-
    DEP_normCounts@colData$Cell.Line[match(counts_data_list_goi[[goi[i]]]$sample_id,
                                          rownames(DEP_normCounts@colData))]
  counts_data_list_goi[[goi[i]]]$drug <-
    DEP_normCounts@colData$drug[match(counts_data_list_goi[[goi[i]]]$sample_id,
                                       rownames(DEP_normCounts@colData))]
  counts_data_list_goi[[goi[i]]]$drug_conc_uM <-
    DEP_normCounts@colData$drug_conc_uM[match(counts_data_list_goi[[goi[i]]]$sample_id,
                                  rownames(DEP_normCounts@colData))]
    counts_data_list_goi[[goi[i]]]$time_h <-
    DEP_normCounts@colData$Timepoint[match(counts_data_list_goi[[goi[i]]]$sample_id,
                                  rownames(DEP_normCounts@colData))]
    counts_data_list_goi[[goi[i]]]$condition <-
    DEP_normCounts@colData$condition[match(counts_data_list_goi[[goi[i]]]$sample_id,
                                  rownames(DEP_normCounts@colData))]
}

# Save count data for GOI
for (i in seq_along(counts_data_list_goi)) {
  write.csv(
  counts_data_list_goi[[i]],
  file = paste0(output_dir_normCounts, names(counts_data_list_goi)[i], ".csv"),
  row.names = TRUE
  )
}

# Compute stats for GOI
counts_data_list_goi_stats <- list()
for (i in seq_along(counts_data_list_goi)) {
  counts_data_list_goi_stats[[names(counts_data_list_goi)[i]]] <-
    counts_data_list_goi[[i]] %>%
    group_by(time_h) %>%
    summarise(
      n = n(),
      avg_normCount = mean(norm_counts),
      sd = sd(norm_counts),
      IQR = IQR(norm_counts),
      MAD = mad(norm_counts),
      Coeff.Variation.Prcnt =sd(norm_counts) / mean(norm_counts) * 100
      )
}

# Save stats data for GOI
for (i in seq_along(counts_data_list_goi_stats)) {
  write.csv(
  counts_data_list_goi_stats[[i]],
  file = paste0(output_dir_normCounts, names(counts_data_list_goi_stats)[i], "_stats.csv"),
  row.names = TRUE
  )
}

# Boxplots on linear scale
b <- list()
for (i in seq_along(counts_data_list_goi)) {
  b[[i]] <- ggplot(data = counts_data_list_goi[[i]],
  aes(x = factor(counts_data_list_goi[[i]]$condition, levels = mixedsort(unique(counts_data_list_goi[[i]]$condition))),
  y = counts_data_list_goi[[i]]$norm_counts)) +
  geom_boxplot() +
  geom_point() +
  xlab("Genotype") +
  ylab("Normalized Counts") +
  scale_y_continuous() +
  labs(title = paste0(names(counts_data_list_goi[i]), " DEP_normCounts")) +
  theme(axis.text.x = element_text(angle = 90, hjust = 0.5, vjust = 0.5, size=rel(1)))
  print(b[[i]])
  ggsave(filename = paste0(
    graph_dir_normCounts,
    names(counts_data_list_goi)[[i]],
    "_normCounts",
    ".pdf"
  ))
}

# Boxplots on linear scale
b <- list()
for (i in seq_along(counts_data_list_goi)) {
  b[[i]] <- ggplot(data = counts_data_list_goi[[i]],
  aes(x = counts_data_list_goi[[i]]$condition ,
  y = counts_data_list_goi[[i]]$norm_counts)) +
  geom_boxplot() +
  geom_point() +
  xlab("Genotype") +
  ylab("Normalized Counts") +
  scale_y_continuous() +
  labs(title = paste0(names(counts_data_list_goi[i]), " DEP_normCounts")) +
  theme(axis.text.x = element_text(angle = 90, hjust = 0.5, vjust = 0.5, size=rel(1)))
  print(b[[i]])
  ggsave(filename = paste0(
    graph_dir_normCounts,
    names(counts_data_list_goi)[[i]],
    "_normCounts",
    ".pdf"
  ))
}

# Graphics termination
if(!is.null(dev.list())) dev.off()
if(!is.null(dev.list())) graphics.off()

})

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##     user   system  elapsed 
##  987.934    2.991 1074.034

This chunk will extract the Impute counts for GOI ##### normCounts (MV)

#  user  system elapsed 
# 602.754   5.045 732.333 

system.time(for (h in seq_along(DEP_TMT_EDA_mv_ls)){

# Directories for normCounts
dir.create(paste0(output_dir,names(DEP_TMT_EDA_mv_ls)[h],"/","normCounts_MV"))
output_dir_normCounts <- paste0(output_dir,names(DEP_TMT_EDA_mv_ls)[h],"/","normCounts_MV/")
dir.create(paste0(graph_dir,names(DEP_TMT_EDA_mv_ls)[h],"/","normCounts_MV"))
graph_dir_normCounts <- paste0(graph_dir,names(DEP_TMT_EDA_mv_ls)[h],"/","normCounts_MV/")

DEP_normCounts <- DEP_TMT_EDA_mv_ls[[h]]

# DEP_normCounts
norm_counts <- as.data.frame(assay(DEP_normCounts))
normCounts <- norm_counts

# Import DF of GOI
# normCounts_goi_df <- read.csv("/Data/Input/RNAseq/REF/normCounts_goi_mouse.csv",header = TRUE, fileEncoding = "UTF-8-BOM")

# vectors of genes of interest. Filtered for present
goi <- row.names(DEP_normCounts)

# Check all goi are present after filtering
match(goi,row.names(normCounts))

# Transform data
counts_goi <- as.data.frame(t(normCounts[goi,]))
counts.rn_counts_goi <- rownames(counts_goi)

# Extract each gene in to a unique dataframe
counts_data_list_goi <- list()
for (i in seq_along(goi)) {
  counts_data_list_goi[[goi[i]]] <- as.data.frame(counts_goi[, i])
  rownames(counts_data_list_goi[[goi[i]]]) <- counts.rn_counts_goi
  counts_data_list_goi[[goi[i]]] <-
    rownames_to_column(counts_data_list_goi[[goi[i]]], var = "sample_id")
  colnames(counts_data_list_goi[[goi[i]]])[2] <- "norm_counts"
  counts_data_list_goi[[goi[i]]]$cell_line_abbr <-
    DEP_normCounts@colData$Cell.Line[match(counts_data_list_goi[[goi[i]]]$sample_id,
                                          rownames(DEP_normCounts@colData))]
  counts_data_list_goi[[goi[i]]]$drug <-
    DEP_normCounts@colData$drug[match(counts_data_list_goi[[goi[i]]]$sample_id,
                                       rownames(DEP_normCounts@colData))]
  counts_data_list_goi[[goi[i]]]$drug_conc_uM <-
    DEP_normCounts@colData$drug_conc_uM[match(counts_data_list_goi[[goi[i]]]$sample_id,
                                  rownames(DEP_normCounts@colData))]
    counts_data_list_goi[[goi[i]]]$time_h <-
    DEP_normCounts@colData$Timepoint[match(counts_data_list_goi[[goi[i]]]$sample_id,
                                  rownames(DEP_normCounts@colData))]
    counts_data_list_goi[[goi[i]]]$condition <-
    DEP_normCounts@colData$condition[match(counts_data_list_goi[[goi[i]]]$sample_id,
                                  rownames(DEP_normCounts@colData))]
}

# Save count data for GOI
for (i in seq_along(counts_data_list_goi)) {
  write.csv(
  counts_data_list_goi[[i]],
  file = paste0(output_dir_normCounts, names(counts_data_list_goi)[i], ".csv"),
  row.names = TRUE
  )
}

# Compute stats for GOI
counts_data_list_goi_stats <- list()
for (i in seq_along(counts_data_list_goi)) {
  counts_data_list_goi_stats[[names(counts_data_list_goi)[i]]] <-
    counts_data_list_goi[[i]] %>%
    group_by(time_h) %>%
    summarise(
      n = n(),
      avg_normCount = mean(norm_counts),
      sd = sd(norm_counts),
      IQR = IQR(norm_counts),
      MAD = mad(norm_counts),
      Coeff.Variation.Prcnt =sd(norm_counts) / mean(norm_counts) * 100
      )
}

# Save stats data for GOI
for (i in seq_along(counts_data_list_goi_stats)) {
  write.csv(
  counts_data_list_goi_stats[[i]],
  file = paste0(output_dir_normCounts, names(counts_data_list_goi_stats)[i], "_stats.csv"),
  row.names = TRUE
  )
}

# Boxplots on linear scale
b <- list()
for (i in seq_along(counts_data_list_goi)) {
  b[[i]] <- ggplot(data = counts_data_list_goi[[i]],
  aes(x = factor(counts_data_list_goi[[i]]$condition, levels = mixedsort(unique(counts_data_list_goi[[i]]$condition))),
  y = counts_data_list_goi[[i]]$norm_counts)) +
  geom_boxplot() +
  geom_point() +
  xlab("Genotype") +
  ylab("Normalized Counts") +
  scale_y_continuous() +
  labs(title = paste0(names(counts_data_list_goi[i]), " DEP_normCounts")) +
  theme(axis.text.x = element_text(angle = 90, hjust = 0.5, vjust = 0.5, size=rel(1)))
  print(b[[i]])
  ggsave(filename = paste0(
    graph_dir_normCounts,
    names(counts_data_list_goi)[[i]],
    "_normCounts",
    ".pdf"
  ))
}

# Boxplots on linear scale
b <- list()
for (i in seq_along(counts_data_list_goi)) {
  b[[i]] <- ggplot(data = counts_data_list_goi[[i]],
  aes(x = counts_data_list_goi[[i]]$condition ,
  y = counts_data_list_goi[[i]]$norm_counts)) +
  geom_boxplot() +
  geom_point() +
  xlab("Genotype") +
  ylab("Normalized Counts") +
  scale_y_continuous() +
  labs(title = paste0(names(counts_data_list_goi[i]), " DEP_normCounts")) +
  theme(axis.text.x = element_text(angle = 90, hjust = 0.5, vjust = 0.5, size=rel(1)))
  print(b[[i]])
  ggsave(filename = paste0(
    graph_dir_normCounts,
    names(counts_data_list_goi)[[i]],
    "_normCounts",
    ".pdf"
  ))
}

# Graphics termination
if(!is.null(dev.list())) dev.off()
if(!is.null(dev.list())) graphics.off()

})

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##     user   system  elapsed 
##  998.139    2.782 1097.893

Limma

Comparisons

comps_ls <- list()
for (h in seq_along(DEP_TMT_EDA_mv_ls)){

# Directories 
dir.create(paste0(output_dir,names(DEP_TMT_EDA_mv_ls)[[h]],"/","Limma_res"))
output_dir_res <- paste0(output_dir,names(DEP_TMT_EDA_mv_ls)[[h]],"/","Limma_res/")
dir.create(paste0(graph_dir,names(DEP_TMT_EDA_mv_ls)[[h]],"/","Limma_res"))
graph_dir_res <- paste0(graph_dir,names(DEP_TMT_EDA_mv_ls)[[h]],"/","Limma_res/")

# Extract one
DEP_mv <- DEP_TMT_EDA_mv_ls[[h]]

# comps
comps_factor <- as.vector(factor(paste0(colData(DEP_mv)$condition)))
comps_df <- crossing(comp_num=comps_factor, comp_denom=comps_factor)
comps_df <- subset(comps_df, as.character(comps_df$comp_num) != as.character(comps_df$comp_denom))

# All comps
comps_df_all <- comps_df
comps_df_all <- cbind(comp_type="condition",comps_df_all)
comps_df_all <- cbind(comp_no=1:nrow(comps_df_all),comps_df_all)
comps_df_all$comp_name <- paste(comps_df_all$comp_num, "vs", comps_df_all$comp_denom, sep = "-")
comps_df_all$comp <- paste(comps_df_all$comp_num,comps_df_all$comp_denom, sep="-")
write.csv(comps_df_all, file=paste0(output_dir_res,"comps_all.csv"), col.names = T, row.names = F)
comps_all_subset <- comps_df_all[,2:4]

# Unique comps only (for only unique combo)
comps_df_unique <- comps_df[!duplicated(t(apply(comps_df, 1, sort))), ]
comps_df_unique <- cbind(comp_type="condition",comps_df_unique )
comps_df_unique <- cbind(comp_no=1:nrow(comps_df_unique),comps_df_unique)
comps_df_unique$comp_name <- paste(comps_df_unique$comp_num, "vs", comps_df_unique$comp_denom, sep = "-")
comps_df_unique$comp <- paste(comps_df_unique$comp_num,comps_df_unique$comp_denom, sep="-")
write.csv(comps_df_unique, file=paste0(output_dir_res,"comps_unique.csv"), col.names = T, row.names = F)
comps_unique_subset <- comps_df_unique[,2:4]

# Comp list to use (all comps)
comps <- comps_df_all

# List of comps
comps_ls[[names(DEP_TMT_EDA_mv_ls)[h]]] <- comps
}

## Warning in write.csv(comps_df_all, file = paste0(output_dir_res,
## "comps_all.csv"), : attempt to set 'col.names' ignored

## Warning in write.csv(comps_df_unique, file = paste0(output_dir_res,
## "comps_unique.csv"), : attempt to set 'col.names' ignored

## Warning in write.csv(comps_df_all, file = paste0(output_dir_res,
## "comps_all.csv"), : attempt to set 'col.names' ignored

## Warning in write.csv(comps_df_unique, file = paste0(output_dir_res,
## "comps_unique.csv"), : attempt to set 'col.names' ignored

## Warning in write.csv(comps_df_all, file = paste0(output_dir_res,
## "comps_all.csv"), : attempt to set 'col.names' ignored

## Warning in write.csv(comps_df_unique, file = paste0(output_dir_res,
## "comps_unique.csv"), : attempt to set 'col.names' ignored

## Warning in write.csv(comps_df_all, file = paste0(output_dir_res,
## "comps_all.csv"), : attempt to set 'col.names' ignored

## Warning in write.csv(comps_df_unique, file = paste0(output_dir_res,
## "comps_unique.csv"), : attempt to set 'col.names' ignored

## Warning in write.csv(comps_df_all, file = paste0(output_dir_res,
## "comps_all.csv"), : attempt to set 'col.names' ignored

## Warning in write.csv(comps_df_unique, file = paste0(output_dir_res,
## "comps_unique.csv"), : attempt to set 'col.names' ignored

## Warning in write.csv(comps_df_all, file = paste0(output_dir_res,
## "comps_all.csv"), : attempt to set 'col.names' ignored

## Warning in write.csv(comps_df_unique, file = paste0(output_dir_res,
## "comps_unique.csv"), : attempt to set 'col.names' ignored

## Warning in write.csv(comps_df_all, file = paste0(output_dir_res,
## "comps_all.csv"), : attempt to set 'col.names' ignored

## Warning in write.csv(comps_df_unique, file = paste0(output_dir_res,
## "comps_unique.csv"), : attempt to set 'col.names' ignored

## Warning in write.csv(comps_df_all, file = paste0(output_dir_res,
## "comps_all.csv"), : attempt to set 'col.names' ignored

## Warning in write.csv(comps_df_unique, file = paste0(output_dir_res,
## "comps_unique.csv"), : attempt to set 'col.names' ignored

## Warning in write.csv(comps_df_all, file = paste0(output_dir_res,
## "comps_all.csv"), : attempt to set 'col.names' ignored

## Warning in write.csv(comps_df_unique, file = paste0(output_dir_res,
## "comps_unique.csv"), : attempt to set 'col.names' ignored

## Warning in write.csv(comps_df_all, file = paste0(output_dir_res,
## "comps_all.csv"), : attempt to set 'col.names' ignored

## Warning in write.csv(comps_df_unique, file = paste0(output_dir_res,
## "comps_unique.csv"), : attempt to set 'col.names' ignored

## Warning in write.csv(comps_df_all, file = paste0(output_dir_res,
## "comps_all.csv"), : attempt to set 'col.names' ignored

## Warning in write.csv(comps_df_unique, file = paste0(output_dir_res,
## "comps_unique.csv"), : attempt to set 'col.names' ignored

DE results

# Extract
# Results
# mibs_comp_list
DEP_TMT_EDA_limmaRes <- list()
for (h in seq_along(comps_ls)){
  
comps <- comps_ls[[h]]

# Directories 
dir.create(paste0(output_dir,names(DEP_TMT_EDA_mv_ls)[[h]],"/","Limma_res"))
output_dir_res <- paste0(output_dir,names(DEP_TMT_EDA_mv_ls)[[h]],"/","Limma_res/")
dir.create(paste0(graph_dir,names(DEP_TMT_EDA_mv_ls)[[h]],"/","Limma_res"))
graph_dir_res <- paste0(graph_dir,names(DEP_TMT_EDA_mv_ls)[[h]],"/","Limma_res/")

# Extract results
DEP_TMT_limmaRes <- list()
for (i in seq_along(comps$comp)){
  DEP_TMT_limmaRes[[comps$comp[i]]] <-
    mibs_comp_list[[paste0(comps$comp[i])]]
}

# save results
for (i in seq_along(DEP_TMT_limmaRes)) {
  write.csv(
  DEP_TMT_limmaRes[[i]],
  file = paste0(output_dir_res,"comp_", i, "_", names(DEP_TMT_limmaRes)[i], ".csv"),
  row.names = TRUE
  )
}
DEP_TMT_EDA_limmaRes[[names(comps_ls)[h]]] <- DEP_TMT_limmaRes
}

## Warning in dir.create(paste0(output_dir, names(DEP_TMT_EDA_mv_ls)[[h]], :
## '/Data/Output/MIB-DYN/DEP_TMT_EDA/C/Limma_res' already exists

## Warning in dir.create(paste0(graph_dir, names(DEP_TMT_EDA_mv_ls)[[h]], "/", :
## '/Data/Graphs/MIB-DYN/DEP_TMT_EDA/C/Limma_res' already exists

## Warning in dir.create(paste0(output_dir, names(DEP_TMT_EDA_mv_ls)[[h]], :
## '/Data/Output/MIB-DYN/DEP_TMT_EDA/CEv3/Limma_res' already exists

## Warning in dir.create(paste0(graph_dir, names(DEP_TMT_EDA_mv_ls)[[h]], "/", :
## '/Data/Graphs/MIB-DYN/DEP_TMT_EDA/CEv3/Limma_res' already exists

## Warning in dir.create(paste0(output_dir, names(DEP_TMT_EDA_mv_ls)[[h]], :
## '/Data/Output/MIB-DYN/DEP_TMT_EDA/G12/Limma_res' already exists

## Warning in dir.create(paste0(graph_dir, names(DEP_TMT_EDA_mv_ls)[[h]], "/", :
## '/Data/Graphs/MIB-DYN/DEP_TMT_EDA/G12/Limma_res' already exists

## Warning in dir.create(paste0(output_dir, names(DEP_TMT_EDA_mv_ls)[[h]], :
## '/Data/Output/MIB-DYN/DEP_TMT_EDA/G8/Limma_res' already exists

## Warning in dir.create(paste0(graph_dir, names(DEP_TMT_EDA_mv_ls)[[h]], "/", :
## '/Data/Graphs/MIB-DYN/DEP_TMT_EDA/G8/Limma_res' already exists

## Warning in dir.create(paste0(output_dir, names(DEP_TMT_EDA_mv_ls)[[h]], :
## '/Data/Output/MIB-DYN/DEP_TMT_EDA/G5/Limma_res' already exists

## Warning in dir.create(paste0(graph_dir, names(DEP_TMT_EDA_mv_ls)[[h]], "/", :
## '/Data/Graphs/MIB-DYN/DEP_TMT_EDA/G5/Limma_res' already exists

## Warning in dir.create(paste0(output_dir, names(DEP_TMT_EDA_mv_ls)[[h]], :
## '/Data/Output/MIB-DYN/DEP_TMT_EDA/G1/Limma_res' already exists

## Warning in dir.create(paste0(graph_dir, names(DEP_TMT_EDA_mv_ls)[[h]], "/", :
## '/Data/Graphs/MIB-DYN/DEP_TMT_EDA/G1/Limma_res' already exists

## Warning in dir.create(paste0(output_dir, names(DEP_TMT_EDA_mv_ls)[[h]], :
## '/Data/Output/MIB-DYN/DEP_TMT_EDA/E5/Limma_res' already exists

## Warning in dir.create(paste0(graph_dir, names(DEP_TMT_EDA_mv_ls)[[h]], "/", :
## '/Data/Graphs/MIB-DYN/DEP_TMT_EDA/E5/Limma_res' already exists

## Warning in dir.create(paste0(output_dir, names(DEP_TMT_EDA_mv_ls)[[h]], :
## '/Data/Output/MIB-DYN/DEP_TMT_EDA/E4/Limma_res' already exists

## Warning in dir.create(paste0(graph_dir, names(DEP_TMT_EDA_mv_ls)[[h]], "/", :
## '/Data/Graphs/MIB-DYN/DEP_TMT_EDA/E4/Limma_res' already exists

## Warning in dir.create(paste0(output_dir, names(DEP_TMT_EDA_mv_ls)[[h]], :
## '/Data/Output/MIB-DYN/DEP_TMT_EDA/TMT_samples_BL_Res/Limma_res' already exists

## Warning in dir.create(paste0(graph_dir, names(DEP_TMT_EDA_mv_ls)[[h]], "/", :
## '/Data/Graphs/MIB-DYN/DEP_TMT_EDA/TMT_samples_BL_Res/Limma_res' already exists

## Warning in dir.create(paste0(output_dir, names(DEP_TMT_EDA_mv_ls)[[h]], :
## '/Data/Output/MIB-DYN/DEP_TMT_EDA/TMT_samples_Res/Limma_res' already exists

## Warning in dir.create(paste0(graph_dir, names(DEP_TMT_EDA_mv_ls)[[h]], "/", :
## '/Data/Graphs/MIB-DYN/DEP_TMT_EDA/TMT_samples_Res/Limma_res' already exists

## Warning in dir.create(paste0(output_dir, names(DEP_TMT_EDA_mv_ls)[[h]], :
## '/Data/Output/MIB-DYN/DEP_TMT_EDA/TMT_samples_Par/Limma_res' already exists

## Warning in dir.create(paste0(graph_dir, names(DEP_TMT_EDA_mv_ls)[[h]], "/", :
## '/Data/Graphs/MIB-DYN/DEP_TMT_EDA/TMT_samples_Par/Limma_res' already exists

Summarize Results

for(h in seq_along(DEP_TMT_EDA_limmaRes)){

comps_df_all <- comps_ls[[names(DEP_TMT_EDA_limmaRes)[h]]]
DEP_TMT_limmaRes <- DEP_TMT_EDA_limmaRes[[h]]

# Directories 
dir.create(paste0(output_dir,names(DEP_TMT_EDA_limmaRes)[[h]],"/","Limma_res"))
output_dir_res <- paste0(output_dir,names(DEP_TMT_EDA_limmaRes)[[h]],"/","Limma_res/")
dir.create(paste0(graph_dir,names(DEP_TMT_EDA_limmaRes)[[h]],"/","Limma_res"))
graph_dir_res <- paste0(graph_dir,names(DEP_TMT_EDA_limmaRes)[[h]],"/","Limma_res/")

# Extract comps of interest
coi <- comps_df_all[grep("0",comps_df_all$comp_denom),]
comp_list_oi <- DEP_TMT_limmaRes[names(DEP_TMT_limmaRes) %in% coi$comp]
comp_list_oi <- lapply(comp_list_oi,data.frame)

# Label list of DF
for (i in seq_along(comp_list_oi)){
  colnames(comp_list_oi[[i]]) <- paste0(colnames(comp_list_oi[[i]]),"_",names(comp_list_oi)[[i]])
  comp_list_oi[[i]]$Gene_ID <- row.names(comp_list_oi[[i]])
}

# Left_join comps of interest to 1x spreadsheet
comp_list_oi_master <- Reduce(function(x, y) left_join(x, y, by="Gene_ID"), comp_list_oi)
row.names(comp_list_oi_master) <- comp_list_oi_master$Gene_ID
comp_list_oi_master <- comp_list_oi_master[,-which(colnames(comp_list_oi_master)=="Gene_ID")]
comp_list_oi_master <- comp_list_oi_master[,-which(colnames(comp_list_oi_master)%in% paste0(grep("gene_",colnames(comp_list_oi_master), value = T)))]


# Subset to spreadsheets
comp_list_oi_padj <- comp_list_oi_master[,grep("adj.P.Val_", colnames(comp_list_oi_master))]
comp_list_oi_pvalue <- comp_list_oi_master[,grep("P.Value_", colnames(comp_list_oi_master))]
comp_list_oi_l2fc <- comp_list_oi_master[,grep("logFC_", colnames(comp_list_oi_master))]

# Save spreadsheets
write.csv(comp_list_oi_padj, file = paste0(output_dir_res,"All_coi_","padj",".csv"))
write.csv(comp_list_oi_pvalue, file = paste0(output_dir_res,"All_coi_","pvalue",".csv"))
write.csv(comp_list_oi_l2fc, file = paste0(output_dir_res,"All_coi_","l2fc",".csv"))
}

## Warning in dir.create(paste0(output_dir, names(DEP_TMT_EDA_limmaRes)[[h]], :
## '/Data/Output/MIB-DYN/DEP_TMT_EDA/C/Limma_res' already exists

## Warning in dir.create(paste0(graph_dir, names(DEP_TMT_EDA_limmaRes)[[h]], :
## '/Data/Graphs/MIB-DYN/DEP_TMT_EDA/C/Limma_res' already exists

## Warning in dir.create(paste0(output_dir, names(DEP_TMT_EDA_limmaRes)[[h]], :
## '/Data/Output/MIB-DYN/DEP_TMT_EDA/CEv3/Limma_res' already exists

## Warning in dir.create(paste0(graph_dir, names(DEP_TMT_EDA_limmaRes)[[h]], :
## '/Data/Graphs/MIB-DYN/DEP_TMT_EDA/CEv3/Limma_res' already exists

## Warning in dir.create(paste0(output_dir, names(DEP_TMT_EDA_limmaRes)[[h]], :
## '/Data/Output/MIB-DYN/DEP_TMT_EDA/G12/Limma_res' already exists

## Warning in dir.create(paste0(graph_dir, names(DEP_TMT_EDA_limmaRes)[[h]], :
## '/Data/Graphs/MIB-DYN/DEP_TMT_EDA/G12/Limma_res' already exists

## Warning in dir.create(paste0(output_dir, names(DEP_TMT_EDA_limmaRes)[[h]], :
## '/Data/Output/MIB-DYN/DEP_TMT_EDA/G8/Limma_res' already exists

## Warning in dir.create(paste0(graph_dir, names(DEP_TMT_EDA_limmaRes)[[h]], :
## '/Data/Graphs/MIB-DYN/DEP_TMT_EDA/G8/Limma_res' already exists

## Warning in dir.create(paste0(output_dir, names(DEP_TMT_EDA_limmaRes)[[h]], :
## '/Data/Output/MIB-DYN/DEP_TMT_EDA/G5/Limma_res' already exists

## Warning in dir.create(paste0(graph_dir, names(DEP_TMT_EDA_limmaRes)[[h]], :
## '/Data/Graphs/MIB-DYN/DEP_TMT_EDA/G5/Limma_res' already exists

## Warning in dir.create(paste0(output_dir, names(DEP_TMT_EDA_limmaRes)[[h]], :
## '/Data/Output/MIB-DYN/DEP_TMT_EDA/G1/Limma_res' already exists

## Warning in dir.create(paste0(graph_dir, names(DEP_TMT_EDA_limmaRes)[[h]], :
## '/Data/Graphs/MIB-DYN/DEP_TMT_EDA/G1/Limma_res' already exists

## Warning in dir.create(paste0(output_dir, names(DEP_TMT_EDA_limmaRes)[[h]], :
## '/Data/Output/MIB-DYN/DEP_TMT_EDA/E5/Limma_res' already exists

## Warning in dir.create(paste0(graph_dir, names(DEP_TMT_EDA_limmaRes)[[h]], :
## '/Data/Graphs/MIB-DYN/DEP_TMT_EDA/E5/Limma_res' already exists

## Warning in dir.create(paste0(output_dir, names(DEP_TMT_EDA_limmaRes)[[h]], :
## '/Data/Output/MIB-DYN/DEP_TMT_EDA/E4/Limma_res' already exists

## Warning in dir.create(paste0(graph_dir, names(DEP_TMT_EDA_limmaRes)[[h]], :
## '/Data/Graphs/MIB-DYN/DEP_TMT_EDA/E4/Limma_res' already exists

## Warning in dir.create(paste0(output_dir, names(DEP_TMT_EDA_limmaRes)[[h]], :
## '/Data/Output/MIB-DYN/DEP_TMT_EDA/TMT_samples_BL_Res/Limma_res' already exists

## Warning in dir.create(paste0(graph_dir, names(DEP_TMT_EDA_limmaRes)[[h]], :
## '/Data/Graphs/MIB-DYN/DEP_TMT_EDA/TMT_samples_BL_Res/Limma_res' already exists

## Warning in dir.create(paste0(output_dir, names(DEP_TMT_EDA_limmaRes)[[h]], :
## '/Data/Output/MIB-DYN/DEP_TMT_EDA/TMT_samples_Res/Limma_res' already exists

## Warning in dir.create(paste0(graph_dir, names(DEP_TMT_EDA_limmaRes)[[h]], :
## '/Data/Graphs/MIB-DYN/DEP_TMT_EDA/TMT_samples_Res/Limma_res' already exists

## Warning in dir.create(paste0(output_dir, names(DEP_TMT_EDA_limmaRes)[[h]], :
## '/Data/Output/MIB-DYN/DEP_TMT_EDA/TMT_samples_Par/Limma_res' already exists

## Warning in dir.create(paste0(graph_dir, names(DEP_TMT_EDA_limmaRes)[[h]], :
## '/Data/Graphs/MIB-DYN/DEP_TMT_EDA/TMT_samples_Par/Limma_res' already exists

Volcano Plots

for(h in seq_along(DEP_TMT_EDA_limmaRes)){
  
# Extract one
subset_limmaRes <- DEP_TMT_EDA_limmaRes[[h]]
  
# Directory for volcano plots
dir.create(paste0(graph_dir,names(DEP_TMT_EDA_limmaRes)[h],"/","volcano"))
graph_dir_vol <- paste0(graph_dir,names(DEP_TMT_EDA_limmaRes)[h],"/","volcano/")

# Plot Volcano
volcano_plots_ls <- list()
for (i in seq_along(subset_limmaRes)){
  plot(volcano_plots_ls[[names(subset_limmaRes)[i]]] <- 
    EnhancedVolcano(
      subset_limmaRes[[i]], 
      lab=row.names(subset_limmaRes[[i]]),
      x="logFC",
      y="adj.P.Val",
      title= paste0("MIBs ",names(subset_limmaRes)[[i]]),
      pCutoff =qval, 
      FCcutoff =lfc,
      pointSize = 2.0, 
      labSize = 3.0 
    ))}

# Save Volcano
for (i in seq_along(volcano_plots_ls)){
ggsave(filename = paste0(graph_dir_vol,"comp_", i, "_", names(volcano_plots_ls)[[i]],".pdf"), plot=volcano_plots_ls[[i]])
}

# Graphics termination 
if(!is.null(dev.list())) dev.off()
if(!is.null(dev.list())) graphics.off()

}

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Session Info

sessionInfo()

## R version 4.3.2 (2023-10-31)
## Platform: x86_64-pc-linux-gnu (64-bit)
## Running under: Ubuntu 22.04.4 LTS
## 
## Matrix products: default
## BLAS:   /usr/lib/x86_64-linux-gnu/openblas-pthread/libblas.so.3 
## LAPACK: /usr/lib/x86_64-linux-gnu/openblas-pthread/libopenblasp-r0.3.20.so;  LAPACK version 3.10.0
## 
## locale:
##  [1] LC_CTYPE=en_US.UTF-8       LC_NUMERIC=C              
##  [3] LC_TIME=en_US.UTF-8        LC_COLLATE=en_US.UTF-8    
##  [5] LC_MONETARY=en_US.UTF-8    LC_MESSAGES=en_US.UTF-8   
##  [7] LC_PAPER=en_US.UTF-8       LC_NAME=C                 
##  [9] LC_ADDRESS=C               LC_TELEPHONE=C            
## [11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C       
## 
## time zone: Etc/UTC
## tzcode source: system (glibc)
## 
## attached base packages:
##  [1] parallel  grid      stats4    stats     graphics  grDevices utils    
##  [8] datasets  methods   base     
## 
## other attached packages:
##  [1] factoextra_1.0.7            cluster_2.1.8              
##  [3] gtools_3.9.5                limma_3.58.1               
##  [5] DEP_1.24.0                  PCAtools_2.14.0            
##  [7] ggpubr_0.6.0                gprofiler2_0.2.3           
##  [9] vsn_3.70.0                  ComplexHeatmap_2.18.0      
## [11] UpSetR_1.4.0                msigdbr_7.5.1              
## [13] GSVA_1.50.5                 RSQLite_2.3.9              
## [15] rmarkdown_2.29              gplots_3.2.0               
## [17] EnhancedVolcano_1.20.0      ggrepel_0.9.6              
## [19] org.Hs.eg.db_3.18.0         AnnotationDbi_1.64.1       
## [21] BiocParallel_1.36.0         lubridate_1.9.4            
## [23] forcats_1.0.0               stringr_1.5.1              
## [25] purrr_1.0.4                 readr_2.1.5                
## [27] tidyr_1.3.1                 tidyverse_2.0.0            
## [29] pheatmap_1.0.12             RColorBrewer_1.1-3         
## [31] colorRamps_2.3.4            cowplot_1.1.3              
## [33] gridExtra_2.3               scales_1.3.0               
## [35] ggplot2_3.5.1               DESeq2_1.42.1              
## [37] SummarizedExperiment_1.32.0 MatrixGenerics_1.14.0      
## [39] matrixStats_1.5.0           tximport_1.30.0            
## [41] Biobase_2.62.0              dplyr_1.1.4                
## [43] biomaRt_2.58.2              rtracklayer_1.62.0         
## [45] GenomicRanges_1.54.1        GenomeInfoDb_1.38.8        
## [47] IRanges_2.36.0              S4Vectors_0.40.2           
## [49] BiocGenerics_0.48.1         tibble_3.2.1               
## 
## loaded via a namespace (and not attached):
##   [1] ProtGenerics_1.34.0         bitops_1.0-9               
##   [3] httr_1.4.7                  doParallel_1.0.17          
##   [5] tools_4.3.2                 MSnbase_2.28.1             
##   [7] backports_1.5.0             DT_0.33                    
##   [9] R6_2.6.1                    HDF5Array_1.30.1           
##  [11] lazyeval_0.2.2              rhdf5filters_1.14.1        
##  [13] GetoptLong_1.0.5            withr_3.0.2                
##  [15] prettyunits_1.2.0           preprocessCore_1.64.0      
##  [17] textshaping_1.0.0           cli_3.6.4                  
##  [19] sandwich_3.1-1              labeling_0.4.3             
##  [21] sass_0.4.9                  mvtnorm_1.3-3              
##  [23] systemfonts_1.2.1           Rsamtools_2.18.0           
##  [25] rstudioapi_0.17.1           impute_1.76.0              
##  [27] generics_0.1.3              shape_1.4.6.1              
##  [29] BiocIO_1.12.0               car_3.1-3                  
##  [31] Matrix_1.6-5                MALDIquant_1.22.3          
##  [33] imputeLCMD_2.1              abind_1.4-8                
##  [35] lifecycle_1.0.4             yaml_2.3.10                
##  [37] carData_3.0-5               rhdf5_2.46.1               
##  [39] SparseArray_1.2.4           BiocFileCache_2.10.2       
##  [41] blob_1.2.4                  promises_1.3.2             
##  [43] dqrng_0.4.1                 crayon_1.5.3               
##  [45] shinydashboard_0.7.2        lattice_0.22-6             
##  [47] beachmat_2.18.1             annotate_1.80.0            
##  [49] mzR_2.36.0                  KEGGREST_1.42.0            
##  [51] pillar_1.10.1               knitr_1.49                 
##  [53] rjson_0.2.23                codetools_0.2-20           
##  [55] glue_1.8.0                  pcaMethods_1.94.0          
##  [57] data.table_1.16.4           vctrs_0.6.5                
##  [59] png_0.1-8                   gtable_0.3.6               
##  [61] assertthat_0.2.1            cachem_1.1.0               
##  [63] xfun_0.51                   mime_0.12                  
##  [65] S4Arrays_1.2.1              ncdf4_1.23                 
##  [67] SingleCellExperiment_1.24.0 iterators_1.0.14           
##  [69] gmm_1.8                     statmod_1.5.0              
##  [71] bit64_4.6.0-1               progress_1.2.3             
##  [73] filelock_1.0.3              bslib_0.9.0                
##  [75] affyio_1.72.0               tmvtnorm_1.6               
##  [77] irlba_2.3.5.1               KernSmooth_2.23-26         
##  [79] colorspace_2.1-1            DBI_1.2.3                  
##  [81] tidyselect_1.2.1            bit_4.5.0.1                
##  [83] compiler_4.3.2              curl_6.2.0                 
##  [85] graph_1.80.0                xml2_1.3.6                 
##  [87] DelayedArray_0.28.0         plotly_4.10.4              
##  [89] caTools_1.18.3              hexbin_1.28.5              
##  [91] affy_1.80.0                 rappdirs_0.3.3             
##  [93] digest_0.6.37               XVector_0.42.0             
##  [95] htmltools_0.5.8.1           pkgconfig_2.0.3            
##  [97] sparseMatrixStats_1.14.0    dbplyr_2.5.0               
##  [99] fastmap_1.2.0               rlang_1.1.5                
## [101] GlobalOptions_0.1.2         htmlwidgets_1.6.4          
## [103] shiny_1.10.0                DelayedMatrixStats_1.24.0  
## [105] farver_2.1.2                jquerylib_0.1.4            
## [107] zoo_1.8-13                  jsonlite_1.9.0             
## [109] mzID_1.40.0                 BiocSingular_1.18.0        
## [111] RCurl_1.98-1.16             magrittr_2.0.3             
## [113] Formula_1.2-5               GenomeInfoDbData_1.2.11    
## [115] Rhdf5lib_1.24.2             munsell_0.5.1              
## [117] Rcpp_1.0.14                 babelgene_22.9             
## [119] MsCoreUtils_1.14.1          stringi_1.8.4              
## [121] zlibbioc_1.48.2             MASS_7.3-60                
## [123] plyr_1.8.9                  Biostrings_2.70.3          
## [125] hms_1.1.3                   circlize_0.4.16            
## [127] locfit_1.5-9.11             ggsignif_0.6.4             
## [129] reshape2_1.4.4              ScaledMatrix_1.10.0        
## [131] XML_3.99-0.18               evaluate_1.0.3             
## [133] BiocManager_1.30.25         httpuv_1.6.15              
## [135] tzdb_0.4.0                  foreach_1.5.2              
## [137] clue_0.3-66                 norm_1.0-11.1              
## [139] rsvd_1.0.5                  broom_1.0.7                
## [141] xtable_1.8-4                restfulr_0.0.15            
## [143] later_1.4.1                 rstatix_0.7.2              
## [145] ragg_1.3.3                  viridisLite_0.4.2          
## [147] memoise_2.0.1               GenomicAlignments_1.38.2   
## [149] timechange_0.3.0            GSEABase_1.64.0

Timing End

proc.time() - ptm

##     user   system  elapsed 
## 2398.887    8.533 2645.174